FLAVONOID AND ANTHOCYANIN BIOPRODUCTION USING MICROORGANISM HOSTS

Application No. 17/720,020

• Filed: Apr 13, 2022
• Examiner: PAK, YONG D
• Art Unit: 1652
• Tech Center: 1600 — Biotechnology & Organic Chemistry
• Assignee: Debut Biotechnology Inc.
• OA Round: 5 (Non-Final)

Office Action

Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .

DETAILED ACTION
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on February 13, 2026 has been entered.

Election/Restrictions
Applicant elected Group I with a species election of (1) E. coli as the host cell, (2) overexpression of cytochrome P450 reductase (CPR) and flavonoid-3’-hydroxylase (F3’H) with N-terminal sequence removed as the genetic modification made in the host cell, (3) dihydrokaempferol (DHK) as the starting material, and (4) dihydroquercetin (DHQ) as the product in the reply filed on June 13, 2023. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 14-15, 19, 21-22, and 24-25 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species/invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on June 13, 2023.

Status of Claims
Claims 1-2, 6, 8-9, 14-15, 19, 21-22, 24-25, and 27-39 are pending.
Claims 14-15, 19, 21-22, and 24-25 are withdrawn.
Claims 1-2, 6, 8-9, and 27-39 are under examination.

Information Disclosure Statement
The information disclosure statement (IDS) submitted on March 19, 2026 and March 24, 2026 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.

Specification
The specification defines “DHM” as dihydromyricein and dihydromyricetin, see page 60. DHM is the acronym for dihydromyricetin, as evidenced by Leonard (Fig. 1, Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab Eng. 2006 Mar;8(2):172-81. Epub 2005 Dec 27. – cited previously on form PTO-892) and Seitz (“Abbreviations:” at page 365 of Plant Mol Biol 61, 365–381 (2006) – cited previously on form PTO-892). Appropriate correction of the expanded phrase of “DHM” is required.

Claim Objections
Claim 1 is objected to because of the following informalities: Claim 1 recites “dihydromyricein (DHM)”. However, DHM is the acronym for dihydromyricetin, as evidenced by Leonard (Fig. 1, Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab Eng. 2006 Mar;8(2):172-81. Epub 2005 Dec 27. – cited previously on form PTO-892) and Seitz (“Abbreviations:” at page 365 of Plant Mol Biol 61, 365–381 (2006) – cited previously on form PTO-892). Appropriate correction of the expanded phrase of “DHM” is required.
For examination purposes, dihydromyricetin is used for the expanded phrase of “DHM”.

Response to Amendment/Arguments

Claim Rejections - 35 USC § 103 – Withdrawn & Maintained
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.

The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.

Withdrawn Rejection
Applicant's arguments, see pages 7-8 of the Remarks, filed June 26, 2025, with respect to claims 1-2, 6, 8-9, and 27 have been fully considered and are persuasive. Claim 1 has been amended to require a dihydroflavonol 4-reductase (DFR) having at least 95% sequence identity to SEQ ID NO:11, 58, 59, 60, or 61, which is not taught by Xu, Leonard, de Vetten, and Seitz. Therefore, the rejection of claims 1-2, 6, 8-9, and 27 under 35 U.S.C. 103 as being unpatentable over Xu (Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus. J Plant Physiol. 2007 Mar;164(3):350-63 – cited previously on form PTO-892), Leonard (Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab Eng. 2006 Mar;8(2):172-81. Epub 2005 Dec 27. – cited previously on form PTO-892), de Vetten (A cytochrome b5 is required for full activity of flavonoid 3', 5'-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):778-83 – cited previously on form PTO-892), and Seitz (Plant Mol Biol 61, 365–381 (2006) – cited previously on form PTO-892) has been withdrawn.

Maintained/Modified Rejection
Claims 1-2, 6, 8-9, 27, and 35 is/are rejected under 35 U.S.C. 103 as being unpatentable over Naesby (“Evolva SA” WO 2017/050853 – form PTO-1449), Xu (Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus. J Plant Physiol. 2007 Mar;164(3):350-63 – cited previously on form PTO-892), Seitz (Plant Mol Biol 61, 365–381 (2006) – cited previously on form PTO-892), Leonard (Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab Eng. 2006 Mar;8(2):172-81. Epub 2005 Dec 27. – cited previously on form PTO-892), and de Vetten (A cytochrome b5 is required for full activity of flavonoid 3', 5'-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):778-83 – cited previously on form PTO-892).
Naesby maps out the biosynthetic route for the production of anthrocyanins, which includes production of dihydrokaempferol (DHK), dihydroquercetin (DHQ), dihydromyricetin (DHM), leucopelargonidin, leucocyanidin, and Leucodelphinidin (Fig. 1 and [0014]). Naesby discloses that anthrocyanins are useful as dyes or coloring agents ([0055]). Naesby discloses that naringenin is converted to (a) DHK, (b) DHQ, and (c) DHM by (a) F3H, (b) F3H, F3’H, and (c) F3H, F3’5’H, respectively (FIG. 1). Naesby discloses that (a) DHK, (b) DHQ, and (c) DHM is converted to leucopelargonidin, leucocyanidin, and Leucodelphinidin, respectively, by DFR (FIG. 1).
Regarding claims 1, 27 and 35, Naesby discloses an engineered E. coli to increase production of DHK, DHQ, and DHM comprising (1) cytochrome P450 reductase (CPR), (2) flavanone 3-hydroxylase (F3H), flavonoid 3’-hydroxylase (F3’H), and/or flavonoid 3',5'-hydroxylase (F3’5’H), and (3) dihydroflavonol 4-reductase (DFR) having the amino acid sequence of SEQ ID NO:6 encoded by SEQ ID NO:5 and 4CL, CHS, CHI, PAL, TAL, and C4H (Fig. 1, [0003], [0068], [00206], and claims 1, 3, 9, and 14). Naesby discloses that E. coli does not naturally produce anthrocyanin ([0057]). Therefore, the engineered E. coli of Naesby has increased production of anthrocyanins and its precurors, DHK, DHQ, and DHM. The DFR of SEQ ID NO:6 of Naesby has 100% sequence identity to the DFR of SEQ ID NO:11 of the instant application (see the sequence alignment below).
Regarding claim 2, Naesby discloses that the precursor for DHQ, DHM is naringenin and/or DHK (Fig. 1 and [0068]).
Naesby does not disclose an engineered E. coli comprising F3’H having the amino acid sequence of SEQ ID NO:8, CPR having the amino acid sequence of SEQ ID NO:9, F3’H fused with CPR, and cytochrome b2 having the amino acid sequence of SEQ ID NO:98.

Regarding claims 1, 8, and 27, Xu discloses an engineered E. coli overexpressing Brassica napus F3’H having 3’-hdyroxylation activity (abstract,4th paragraph at page 353, and 2nd full paragraph at page 360). The Brassica napus F3’H of Xu has 100% sequence identity to the Brassica napus F3’H of SEQ ID NO:8 of the instant application (Figure 1 at page 355 and see the sequence alignment below). Brassica napus F3’H of Xu has an N-terminal membrane anchoring domain, residues 1-22 (Figure 1 and 3rd-4th full paragraphs at page 360).
Regarding claim 1, Seitz discloses that F3’Hs catalyze hydroxylation at the 3’-position of the B ring of flavonoids and F3’Hs have been shown to hydroxylate a broad range of flavonoid substrates including DHK (2nd full paragraph at page 366, Table 1 at page 367, and Figure 1 at page 371).

Regarding claims 1, 6, 9, and 27, Leonard discloses an engineered E. coli overexpressing 4CL, CHS, CHI, FHT, FLS, and CPR-F3’5’H soluble fusion protein (Section 2.4 at pages 174-175, Table 2 at page 175, Section 3.1 at pages 175-176, Table 3 at page 179, and Figure 5 at page 179). Said engineered E. coli produces DHQ, and DHM (Fig 1 at page 173, top right paragraph at page 173, and Table 2 at page 175). Leonard discloses that F3’H and F3’5’H both hydroxylate the 3’-position of the B ring of DHK to produce DHQ (Fig. 1). Leonard discloses that the N-terminal leader sequence/membrane anchoring domain of the F3’5H has been removed to facilitation function expression of F3’5’H (Section 3.1 at pages 175-176). Leonard discloses that in order to achieve functional expression of F3’5’H, truncated F3’5’H was fused with its redox partner CPR (1st full paragraph at page 174). The CPR of Leonard is a C. roseus having GenBank Accession number X69791 (see top paragraph at page 174). C. roseus CPR having GenBank Accession number X69791 has 100% sequence identity to the C. roseus CPR of SEQ D NO:9 of the instant application (see the sequence alignment below). Leonard discloses a truncated CPR (N-terminal leader sequence of 69 amino acids) (Section 3.1 at pages 175-176).

Regarding claim 1, de Vetten discloses that cytochrome b5 is required for full activity of F3',5'H (abstract). de Vetten discloses cDNA encoding a cytochrome b5 having 100% sequence identity to the cytochrome b5 of SEQ ID NO:98 of the instant application (page 779 right column and see the sequence alignment below).

Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art at the time the claimed invention was effectively filed to modify the engineered E. coli of Naesby by expressing truncated B. napus F3’H (which is identical to SEQ ID NO:8 of the instant application) fused to truncated C. roseus CPR (which is identical to SEQ ID NO:9) in combination with 4CL, CHS, CHI, PAL, TAL, C4H, DFR (identical to SEQ ID NO:11), and CPR-F3’5’H and also express cytochrome b5 (which is identical to SEQ ID NO:98) of de Vetten. One having ordinary skill in the art would have been motivated to express truncated B. napus F3’H fused to truncated C. roseus CPR in combination with 4CL, CHS, CHI, PAL, TAL, C4H and CPR-F3’5’H and also express cytochrome b5 of de Vetten in order to increase DHK, DHQ, and DHM and leucopelargonidin, leucocyanidin, and Leucodelphinidin production in E. coli. One having ordinary skill in the art would have been motivated to remove the N-terminal membrane anchoring domain of the B. napus F3’H of Xu and fuse the truncated F3’H to the truncated C. roseus CPR, redox partner, to express a functional soluble F3’H similar to F3’5’H since both F3’H and F3’5’H have N-terminal membrane anchoring domains. One having ordinary skill in the art would have been motivated to express cytochrome b5 in order to ensure full activity of the F’3’5’H since cytochrome b5 is required for full activity of F3’5’H. One of ordinary skill in the art would have had a reasonable expectation of success since Naesby discloses an engineered E. coli having increased production of (DHK), dihydroquercetin (DHQ), dihydromyricetin (DHM), leucopelargonidin, leucocyanidin, and Leucodelphinidin, wherein the E. coli expresses CPR, F3H, F3’H, F3’5’H, and DFR in combination with 4CL, CHS, CHI, PAL, TAL, and C4H, Xu discloses E. coli engineered to express B. napus F3’H, Leonard discloses biosynthesis DHQ in an engineered E. coli, Leonard discloses that F3’H converts DHK to DHQ, de Vetten discloses cDNA encoding a cytochrome b5 that is required for full activity of F3’5’H, and Seitz discloses that F3’Hs catalyze hydroxylation at the 3’-position of the B ring of flavonoids and F3’Hs have been shown to hydroxylate a broad range of flavonoid substrates including DHK.
Therefore, the above references render claims 1-2, 6, 8-9, 27, and 35 prima facie obvious. Applicant should note that the rejection has been amended in light of the amendment of the claims.
Applicant's arguments filed February 13, 2026 have been fully considered but they are not persuasive.
Applicant argues that claims are not obvious over Xu, Leonard, de Vetten, and Seitz and one having ordinary skill in the art would not have been motivated to the combine the above cited references because (A) although Xu discloses Brassica napus F3’H having 100% sequence identity to SEQ ID NO:8 of the instant application, Xu does not disclose or suggest an engineered E. coli host cell expressing F3’H, much less with CPR or cytochrome b5 as recited in claim 1, (B) although Leonard discloses engineering E. coli to produce kaempferol and quercetin by simultaneous co-expressing a F3’5’H-CPR in combination with five additional components of 4CL, CHS, CHI, FHT, and FLS, F3’H and F3’5’H are different enzymes. Applicant argues that F3’5’H can act on both DHK and DHQ (converting them to DHM) while F3’H specifically catalyzes the formation of DHQ from DHK and Leonard is limited to F3’5’H as a fusion protein with a P450 reductase, (C) none of the references provide F3’H as a fusion with CPR, (D) although de Vetten discloses that cytochrome b5 is required for full activity of F3’5’H, de Vetten and the cited references do not provide showing that cytochrome b5 is required for full activity of F3’H, and (E) although Seitz discloses that F3’H hydroxylates the 3’-position of the B ring of flavonoids including DHK, F3’H and F3’5’H are substantially different enzymes.
This is not found persuasive.
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Further, the claims do not exclude expression of other enzymes, such as 4CL, CHS, CHI, PAL, TAL, C4H, and CPR-F3’5’H in an E. coli engineered host cell comprising CPR-F3’H, cytochrome b5, and DFR.
(A)(i) and (C) Brassica napus F3’H of Xu has an N-terminal membrane anchoring domain, residues 1-22 (Figure 1 and 3rd-4th full paragraphs at page 360). Leonard discloses that the N-terminal leader sequence/membrane anchoring domain of the F3’5H has been removed to facilitation function expression of F3’5’H (Section 3.1 at pages 175-176). Leonard discloses that in order to achieve functional expression of F3’5’H, truncated F3’5’H was fused with its redox partner CPR (1st full paragraph at page 174). Therefore, one having ordinary skill in the art would have been motivated to remove the N-terminal membrane anchoring domain of the B. napus F3’H of Xu and fuse the truncated F3’H to the truncated C. roseus CPR, redox partner, to express a functional soluble F3’H similar to F3’5’H since both F3’H and F3’5’H have N-terminal membrane anchoring domains. (ii) One having ordinary skill in the art would have been motivated to express cytochrome b5 in order to ensure full activity of the F’3’5’H since cytochrome b5 is required for full activity of F3’5’H. F3’5’H catalyzes conversion of naringenin to DHM (Fig 1 of Naesby) and catalyzes conversion of DHK to DHM (Fig. 1 of Leonard).
(B) and (E) The rejection does not assert that F3’H and F3’5’H have the same enzymatic activity. The claims do not exclude expression of F3’5’H. In combining the cited references, it would have been obvious to express expressing truncated B. napus F3’H fused to truncated C. roseus CPR in combination with 4CL, CHS, CHI, PAL, TAL, C4H, DFR and CPR-F3’5’H and also express cytochrome b5 in order to increase DHQ, DHK, and DHM production in E. coli.
(D) The rejection does not assert that cytochrome b5 is required for full activity of F3’H. de Vetten is relied for its disclosure that cytochrome b5 is required for full activity of F3’5’H.
Applicant argues that none of Xu, Leonard, de Vetten, and Seitz discloses DFR having at least 95% sequence identity to SEQ ID NO:11, 58, 59, 60, or 61.
Naesby discloses an engineered E. col comprising dihydroflavonol 4-reductase (DFR) having the amino acid sequence of SEQ ID NO:6, which has 100% sequence identity to the DFR of SEQ ID NO:11 of the instant application.
Hence the rejection has been maintained.

New Rejections
Claim(s) 28-31 is/are rejected under 35 U.S.C. 103 as being unpatentable over Naesby (“Evolva SA” WO 2017/050853 – form PTO-1449), Xu (Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus. J Plant Physiol. 2007 Mar;164(3):350-63 – cited previously on form PTO-892), Seitz (Plant Mol Biol 61, 365–381 (2006) – cited previously on form PTO-892), Leonard (Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab Eng. 2006 Mar;8(2):172-81. Epub 2005 Dec 27. – cited previously on form PTO-892), and de Vetten (A cytochrome b5 is required for full activity of flavonoid 3', 5'-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):778-83 – cited previously on form PTO-892) as applied to claims 1-2, 6, 8-9, 27, and 35 above, and further in view of A0A1Q3CVI5 (UniProtKB/TrEMBL Database. December 11, 2019 – form PTO-892), A0A061G4T4 (UniProtKB/TrEMBL Database. December 11, 2019 – form PTO-892), Q38L00 (UniProtKB/TrEMBL Database. December 11, 2019 – form PTO-892), and XP_008791304.2 (GenPept Database. October 17, 2018 – form PTO-892).
The combined teachings of Naesby, Xu, Seitz, Leonard, and de Vetten do not disclose a DFR having the amino acid sequence of SEQ ID NO:49, 50, 51, or 52.

Regarding claims 1 and 28, A0A1Q3CVI5 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:49 of the instant application (see page 1 and see the sequence alignment below).
Regarding claims 1 and 29, A0A061G4T4 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:50 of the instant application (see pages 1-2 and see the sequence alignment below).
Regarding claims 1 and 30, Q38L00 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:51 of the instant application (see page 1 and see the sequence alignment below).
Regarding claims 1 and 31, XP_008791304.2 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:52 of the instant application (see pages 1-2 and see the sequence alignment below).

Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to replace the F3’H of Naesby or Xu with other known F3’H, such as the F3’H of A0A1Q3CVI5, A0A061G4T4, Q38L00 or XP_008791304.2 because one of ordinary skill in the art would have been able to carry out such a substitution, and the results were reasonably predictable. One having ordinary skill in the art would have been motivated to do so in order to further increase and optimize the production DHQ in E. coli. One of ordinary skill in the art would have had a reasonable expectation of success since Naesby discloses an engineered E. coli having increased production of (DHK), dihydroquercetin (DHQ), dihydromyricetin (DHM), leucopelargonidin, leucocyanidin, and Leucodelphinidin, wherein the E. coli expresses CPR, F3H, F3’H, F3’5’H, and DFR in combination with 4CL, CHS, CHI, PAL, TAL, and C4H, Xu discloses E. coli engineered to express B. napus F3’H, Leonard discloses biosynthesis DHQ in an engineered E. coli, Leonard discloses that F3’H converts DHK to DHQ, de Vetten discloses cDNA encoding a cytochrome b5 that is required for full activity of F3’5’H, Seitz discloses that F3’Hs catalyze hydroxylation at the 3’-position of the B ring of flavonoids and F3’Hs have been shown to hydroxylate a broad range of flavonoid substrates including DHK., and A0A1Q3CVI5, A0A061G4T4, Q38L00 and XP_008791304.2 disclose F3’H. The rationale to support a conclusion that the claims would have been obvious is that the substitution of one known element (F3’H of Naesby or Xu) for another (other known F3’Hs) yields predictable results (conversion of naringenin to DHQ). See MPEP 2143.
Therefore, the above references render claims 1-2, 6, 8-9, 27-31, and 35 prima facie obvious.

Claim(s) 32-34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Naesby (“Evolva SA” WO 2017/050853 – form PTO-1449), Xu (Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus. J Plant Physiol. 2007 Mar;164(3):350-63 – cited previously on form PTO-892), Seitz (Plant Mol Biol 61, 365–381 (2006) – cited previously on form PTO-892), Leonard (Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab Eng. 2006 Mar;8(2):172-81. Epub 2005 Dec 27. – cited previously on form PTO-892), and de Vetten (A cytochrome b5 is required for full activity of flavonoid 3', 5'-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):778-83 – cited previously on form PTO-892) as applied to claims 1-2, 6, 8-9, 27, and 35 above, and further in view of XP_028084858.1 (GenPept Database. March 5, 2018 – form PTO-892) and A0A0D3DGY4 (UniProtKB/TrEMBL Database. December 11, 2019– form PTO-892).
The combined teachings of Naesby, Xu, Seitz, Leonard, and de Vetten do not disclose a CPR having the amino acid sequence of SEQ ID NO:53, 54, or 55.

Regarding claims 1 and 32-33, XP_028084858.1 discloses a CPR having 100% sequence identity to the CPR of SEQ ID NO:53 and SEQ ID NO:54 of the instant application (see page 1 and see the sequence alignment below).
Regarding claims 1 and 34, A0A0D3DGY4 discloses a CPR having at least 95 % sequence identity to the CPR of SEQ ID NO:55 of the instant application (see page 1 and see the sequence alignment below).

Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to replace the CPR of Leonard with other known CPR, such as the CPR of XP_028084858.1 or A0A0D3DGY4 because one of ordinary skill in the art would have been able to carry out such a substitution, and the results were reasonably predictable. One having ordinary skill in the art would have been motivated to do so in order to further increase and optimize the production DHK, DHQ, and DHM in E. coli. One of ordinary skill in the art would have had a reasonable expectation of success since Naesby discloses an engineered E. coli having increased production of (DHK), dihydroquercetin (DHQ), dihydromyricetin (DHM), leucopelargonidin, leucocyanidin, and Leucodelphinidin, wherein the E. coli expresses CPR, F3H, F3’H, F3’5’H, and DFR in combination with 4CL, CHS, CHI, PAL, TAL, and C4H, Xu discloses E. coli engineered to express B. napus F3’H, Leonard discloses biosynthesis DHQ in an engineered E. coli, Leonard discloses that F3’H converts DHK to DHQ, de Vetten discloses cDNA encoding a cytochrome b5 that is required for full activity of F3’5’H, Seitz discloses that F3’Hs catalyze hydroxylation at the 3’-position of the B ring of flavonoids and F3’Hs have been shown to hydroxylate a broad range of flavonoid substrates including DHK., and XP_028084858.1 and A0A0D3DGY4 disclose CPR. The rationale to support a conclusion that the claims would have been obvious is that the substitution of one known element (CPR of Leonard) for another (other known CPRs) yields predictable results (production of DHK, DHQ, and DHM). See MPEP 2143.
Therefore, the above references render claims 1-2, 6, 8-9, 27, and 32-35 prima facie obvious.

Claim(s) 36-39 is/are rejected under 35 U.S.C. 103 as being unpatentable over Naesby (“Evolva SA” WO 2017/050853 – form PTO-1449), Xu (Cloning and molecular characterization of a functional flavonoid 3'-hydroxylase gene from Brassica napus. J Plant Physiol. 2007 Mar;164(3):350-63 – cited previously on form PTO-892), Seitz (Plant Mol Biol 61, 365–381 (2006) – cited previously on form PTO-892), Leonard (Functional expression of a P450 flavonoid hydroxylase for the biosynthesis of plant-specific hydroxylated flavonols in Escherichia coli. Metab Eng. 2006 Mar;8(2):172-81. Epub 2005 Dec 27. – cited previously on form PTO-892), and de Vetten (A cytochrome b5 is required for full activity of flavonoid 3', 5'-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors. Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):778-83 – cited previously on form PTO-892) as applied to claims 1-2, 6, 8-9, 27, and 35 above, and further in view of A0A2U8UVV3 (UniProtKB/TrEMBL Database, December 11, 2019. – form PTO-892), XP_027329642.1(GenPept Database, December, 20, 2018 – form PTO-892), F5A636 (UniProtKB/TrEMBL Database, December 11, 2019. – form PTO-892) and A0A2P6PYS2 (UniProtKB/TrEMBL Database, December 11, 2019. – form PTO-892).
The combined teachings of Naesby, Xu, Seitz, Leonard, and de Vetten do not disclose DFR having the amino acid sequence of SEQ ID NO:58, 59, 60, or 61.

Regarding claims 1 and 36, A0A2U8UVV3 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:58 of the instant application (see page 1 and see the sequence alignment below).
Regarding claims 1 and 37, XP_027329642.1 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:59 of the instant application (see pages 1-2 and see the sequence alignment below).
Regarding claims 1 and 38, F5A636 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:60 of the instant application (see page 1 and see the sequence alignment below).
Regarding claims 1 and 39, A0A2P6PYS2 discloses a DFR having 100% sequence identity to the DFR of SEQ ID NO:61 of the instant application (see page 1 and see the sequence alignment below).
Therefore, in combining the above references, it would have been obvious to one having ordinary skill in the art before the time the claimed invention was effectively filed to replace the DFR of Naesby with other known DFRs, such as the DFR of A0A2U8UVV3, XP_027329642.1, F5A636, and A0A2P6PYS2 because one of ordinary skill in the art would have been able to carry out such a substitution, and the results were reasonably predictable. One having ordinary skill in the art would have been motivated to do so in order to further increase and optimize the production of leucopelargonidin, leucocyanidin, and Leucodelphinidin in E. coli. One of ordinary skill in the art would have had a reasonable expectation of success since Ajinmoto teaches a method of producing cysteine from glutathione via cysteine-glycine using a glutaminase and a peptidase in acid conditions and Yin teaches an acid protease. The rationale to support a conclusion that the claims would have been obvious is that the substitution of one known element (DFR of Naesby) for another (other known DRF) yields predictable results (conversion of DHM to leucopelargonidin, leucocyanidin, and Leucodelphinidin) to one of ordinary skill in the art since Naesby discloses an engineered E. coli having increased production of (DHK), dihydroquercetin (DHQ), dihydromyricetin (DHM), leucopelargonidin, leucocyanidin, and Leucodelphinidin, wherein the E. coli expresses CPR, F3H, F3’H, F3’5’H, and DFR in combination with 4CL, CHS, CHI, PAL, TAL, and C4H, Xu discloses E. coli engineered to express B. napus F3’H, Leonard discloses biosynthesis DHQ in an engineered E. coli, Leonard discloses that F3’H converts DHK to DHQ, de Vetten discloses cDNA encoding a cytochrome b5 that is required for full activity of F3’5’H, Seitz discloses that F3’Hs catalyze hydroxylation at the 3’-position of the B ring of flavonoids and F3’Hs have been shown to hydroxylate a broad range of flavonoid substrates including DHK., and A0A2U8UVV3, XP_027329642.1, F5A636, and A0A2P6PYS2 disclose DFR. See MPEP 2143.
Therefore, the above references render claims 1-2, 6, 8-9, 27, and 35-39 prima facie obvious.

Duplicate Claims, Warning
Applicant is advised that should claim 32 be found allowable, claim 34 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
The CPR having the amino acid sequence of SEQ ID NO:53 is identical to the CPR having the amino acid sequence of SEQ ID NO:54, see the sequence alignment below.

Conclusion

Claims 1-2, 6, 8-9, 14-15, 19, 21-22, 24-25, and 27-39 are pending.
Claims 14-15, 19, 21-22, and 24-25 are withdrawn.
Claims 1-2, 6, 8-9, and 27-39 are rejected

Any inquiry concerning this communication or earlier communications from the examiner should be directed to YONG D PAK whose telephone number is [phone]. The examiner can normally be reached M-Th: 5:30 am - 3:30 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert Mondesi can be reached on 408-918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/YONG D PAK/Primary Examiner, Art Unit 1652
Sequence alignment of the DFR of SEQ ID NO:11 of the instant application (“Qy”) and DRF of SEQ ID NO:6 of Naesby (“Db”)

BDU40400
ID BDU40400 standard; protein; 347 AA.
XX
AC BDU40400;
XX
DT 18-MAY-2017 (first entry)
XX
DE A. andraeanum dihydroflavonol-4-reductase (DFR) polypeptide SEQ: 6.
XX
KW DFR protein; anthocyanin; dihydroflavonol-4-reductase; fermentation;
KW genetically engineered microorganism; metabolic engineering; pigment;
KW plant.
XX
OS Anthurium andraeanum.
XX
CC PN WO2017050853-A1.
XX
CC PD 30-MAR-2017.
XX
CC PF 21-SEP-2016; 2016WO-EP072474.
XX
PR 24-SEP-2015; 2015US-0222919P.
XX
CC PA (EVOL-) EVOLVA SA.
XX
CC PI Naesby M, Zokouri Z, Fischer D, Eichenberger M, Hansson A;
XX
DR WPI; 2017-22034W/26.
DR N-PSDB; BDU40399.
XX
CC PT New microorganism comprising an operative metabolic pathway capable of
CC PT producing an anthocyanin from a simple sugar, useful for producing an
CC PT anthocyanin.
XX
CC PS Disclosure; SEQ ID NO 6; 129pp; English.
XX
CC The present invention relates to a novel microorganism comprising an
CC operative metabolic pathway capable of producing an anthocyanin from a
CC simple sugar. The operative metabolic pathway contains: (i) a 4-coumaric
CC acid-CoA ligase (4CL); (ii) a chalcone synthase (CHS); (iii) a flavanone
CC 3-hydroxylase (F3H); (iv) a dihydroflavonol-4-reductase (DFR); (v) an
CC anthocyanidin synthase (ANS); (vi) an anthocyanidin 3-O-glycosyl
CC transferase (A3GT); (vii) a chalcone isomerase (CHI); and at least one
CC of: (a) a tyrosine ammonia lyase (TAL); or (b) a phenylalanine ammonia
CC lyase (PAL) and a trans-cinnamate 4-monooxygenase (C4H). The
CC microorganism herein is genetically modified to exhibit increased
CC tolerance to a precursor, an intermediate, or a product molecule from the
CC operative metabolic pathway. The invention claims: (1) a fermentation
CC vessel comprising the microorganism; and (2) a method for producing the
CC anthocyanin by culturing the microorganism. The present sequence
CC represents an Anthurium andraeanum DFR polypeptide sequence useful in the
CC synthesis of anthocyanin.
XX
SQ Sequence 347 AA;

Query Match 100.0%; Score 1833; Length 347;
Best Local Similarity 100.0%;
Matches 347; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATVRDPSNMKKVKHLLDLPGAANRLTLW 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATVRDPSNMKKVKHLLDLPGAANRLTLW 60

Qy 61 KADLVDEGSFDEPIQGCTGVFHVATPMDFESKDPESEMIKPTIEGMLNVLRSCARASSTV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 KADLVDEGSFDEPIQGCTGVFHVATPMDFESKDPESEMIKPTIEGMLNVLRSCARASSTV 120

Qy 121 RRVVFTSSAGTVSIHEGRRHLYDETSWSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 RRVVFTSSAGTVSIHEGRRHLYDETSWSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK 180

Qy 181 NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPHYSILNPVQFVHLDDLCNAHIF 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPHYSILNPVQFVHLDDLCNAHIF 240

Qy 241 LFECPDAKGRYICSSHDVTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKLTDLG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LFECPDAKGRYICSSHDVTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKLTDLG 300

Qy 301 FEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYATEQLIATGQDNGH 347
|||||||||||||||||||||||||||||||||||||||||||||||
Db 301 FEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYATEQLIATGQDNGH 347 NCPR_CATRO

Sequence alignment of C. roseus CPR of SEQ ID NO:9 (“Qy”) and C. roseus CPR of Leonard (“Db”)

ID NCPR_CATRO Reviewed; 714 AA.
AC Q05001;
DT 01-FEB-1995, integrated into UniProtKB/Swiss-Prot.
DT 01-FEB-1995, sequence version 1.
DT 22-FEB-2023, entry version 119.
DE RecName: Full=NADPH--cytochrome P450 reductase {ECO:0000255|HAMAP-Rule:MF_03212};
DE Short=CPR {ECO:0000255|HAMAP-Rule:MF_03212};
DE Short=P450R {ECO:0000255|HAMAP-Rule:MF_03212};
DE EC=1.6.2.4 {ECO:0000255|HAMAP-Rule:MF_03212};
GN Name=CPR;
OS Catharanthus roseus (Madagascar periwinkle) (Vinca rosea).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC asterids; lamiids; Gentianales; Apocynaceae; Rauvolfioideae; Vinceae;
OC Catharanthinae; Catharanthus.
OX NCBI_TaxID=4058;
RN [1]
RP NUCLEOTIDE SEQUENCE [MRNA].
RX PubMed=8220474; DOI=10.1046/j.1365-313x.1993.04010047.x;
RA Meijer A.H., Lopes Cardoso M.I., Voskuilen J.T., de Waal A., Verpoorte R.,
RA Hoge J.H.C.;
RT "Isolation and characterization of a cDNA clone from Catharanthus roseus
RT encoding NADPH:cytochrome P-450 reductase, an enzyme essential for
RT reactions catalysed by cytochrome P-450 mono-oxygenases in plants.";
RL Plant J. 4:47-60(1993).
RN [2]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA] OF 1-11.
RC STRAIN=cv. Morning mist;
RA Lopes Cardoso M.T., Meijer A.H., Rueb S., Queiroz Machado J., Memelink J.,
RA Hoge J.H.C.;
RL Submitted (NOV-1996) to the EMBL/GenBank/DDBJ databases.
CC -!- FUNCTION: This enzyme is required for electron transfer from NADP to
CC cytochrome P450 in microsomes. It can also provide electron transfer to
CC heme oxygenase and cytochrome B5. {ECO:0000255|HAMAP-Rule:MF_03212}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=NADPH + 2 oxidized [cytochrome P450] = H(+) + NADP(+) + 2
CC reduced [cytochrome P450]; Xref=Rhea:RHEA:24040, Rhea:RHEA-
CC COMP:14627, Rhea:RHEA-COMP:14628, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:55376, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:60344; EC=1.6.2.4; Evidence={ECO:0000255|HAMAP-
CC Rule:MF_03212};
CC -!- COFACTOR:
CC Name=FAD; Xref=ChEBI:CHEBI:57692;
CC Evidence={ECO:0000255|HAMAP-Rule:MF_03212};
CC Note=Binds 1 FAD per monomer. {ECO:0000255|HAMAP-Rule:MF_03212};
CC -!- COFACTOR:
CC Name=FMN; Xref=ChEBI:CHEBI:58210;
CC Evidence={ECO:0000255|HAMAP-Rule:MF_03212};
CC Note=Binds 1 FMN per monomer. {ECO:0000255|HAMAP-Rule:MF_03212};
CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane
CC {ECO:0000255|HAMAP-Rule:MF_03212}; Single-pass membrane protein
CC {ECO:0000255|HAMAP-Rule:MF_03212}; Cytoplasmic side {ECO:0000255|HAMAP-
CC Rule:MF_03212}.
CC -!- SIMILARITY: Belongs to the NADPH--cytochrome P450 reductase family.
CC {ECO:0000255|HAMAP-Rule:MF_03212}.
CC -!- SIMILARITY: In the N-terminal section; belongs to the flavodoxin
CC family. {ECO:0000255|HAMAP-Rule:MF_03212}.
CC -!- SIMILARITY: In the C-terminal section; belongs to the flavoprotein
CC pyridine nucleotide cytochrome reductase family. {ECO:0000255|HAMAP-
CC Rule:MF_03212}.
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DR EMBL; X69791; CAA49446.1; -; mRNA.
DR EMBL; Y09417; CAA70571.1; -; Genomic_DNA.
DR PIR; S31502; S31502.
DR AlphaFoldDB; Q05001; -.
DR SMR; Q05001; -.
DR GO; GO:0005789; C:endoplasmic reticulum membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0050660; F:flavin adenine dinucleotide binding; IEA:UniProtKB-UniRule.
DR GO; GO:0010181; F:FMN binding; IEA:UniProtKB-UniRule.
DR GO; GO:0050661; F:NADP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0003958; F:NADPH-hemoprotein reductase activity; IEA:UniProtKB-UniRule.
DR CDD; cd06204; CYPOR; 1.
DR Gene3D; 3.40.50.360; -; 1.
DR Gene3D; 3.40.50.80; Nucleotide-binding domain of ferredoxin-NADP reductase (FNR) module; 1.
DR Gene3D; 2.40.30.10; Translation factors; 1.
DR HAMAP; MF_03212; NCPR; 1.
DR InterPro; IPR003097; CysJ-like_FAD-binding.
DR InterPro; IPR017927; FAD-bd_FR_type.
DR InterPro; IPR001094; Flavdoxin-like.
DR InterPro; IPR008254; Flavodoxin/NO_synth.
DR InterPro; IPR001709; Flavoprot_Pyr_Nucl_cyt_Rdtase.
DR InterPro; IPR029039; Flavoprotein-like_sf.
DR InterPro; IPR039261; FNR_nucleotide-bd.
DR InterPro; IPR023173; NADPH_Cyt_P450_Rdtase_alpha.
DR InterPro; IPR001433; OxRdtase_FAD/NAD-bd.
DR InterPro; IPR023208; P450R.
DR InterPro; IPR017938; Riboflavin_synthase-like_b-brl.
DR PANTHER; PTHR19384:SF17; NADPH--CYTOCHROME P450 REDUCTASE; 1.
DR PANTHER; PTHR19384; NITRIC OXIDE SYNTHASE-RELATED; 1.
DR Pfam; PF00667; FAD_binding_1; 1.
DR Pfam; PF00258; Flavodoxin_1; 1.
DR Pfam; PF00175; NAD_binding_1; 1.
DR PIRSF; PIRSF000208; P450R; 1.
DR PRINTS; PR00369; FLAVODOXIN.
DR PRINTS; PR00371; FPNCR.
DR SUPFAM; SSF52343; Ferredoxin reductase-like, C-terminal NADP-linked domain; 1.
DR SUPFAM; SSF52218; Flavoproteins; 1.
DR SUPFAM; SSF63380; Riboflavin synthase domain-like; 1.
DR PROSITE; PS51384; FAD_FR; 1.
DR PROSITE; PS50902; FLAVODOXIN_LIKE; 1.
PE 2: Evidence at transcript level;
KW Endoplasmic reticulum; FAD; Flavoprotein; FMN; Membrane; NADP;
KW Oxidoreductase; Transmembrane; Transmembrane helix.
FT CHAIN 1..714
FT /note="NADPH--cytochrome P450 reductase"
FT /id="PRO_0000167610"
FT TOPO_DOM 1..48
FT /note="Lumenal"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT TRANSMEM 49..69
FT /note="Helical"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT TOPO_DOM 70..714
FT /note="Cytoplasmic"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT DOMAIN 104..254
FT /note="Flavodoxin-like"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT DOMAIN 312..559
FT /note="FAD-binding FR-type"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 110..115
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 165..168
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 203..212
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 238
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 332
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 492..495
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 510..512
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 526..529
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 573
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 634..635
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 640..644
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 676
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
FT BINDING 714
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_03212"
SQ SEQUENCE 714 AA; 78958 MW; DBDD9AF41374CF91 CRC64;

Query Match 100.0%; Score 3730; Length 714;
Best Local Similarity 100.0%;
Matches 714; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPAVMAMLLENKELVMILTTSVAVL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPAVMAMLLENKELVMILTTSVAVL 60

Qy 61 IGCVVVLIWRRSSGSGKKVVEPPKLIVPKSVVEPEEIDEGKKKFTIFFGTQTGTAEGFAK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 IGCVVVLIWRRSSGSGKKVVEPPKLIVPKSVVEPEEIDEGKKKFTIFFGTQTGTAEGFAK 120

Qy 121 ALAEEAKARYEKAVIKVIDIDDYAADDEEYEEKFRKETLAFFILATYGDGEPTDNAARFY 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ALAEEAKARYEKAVIKVIDIDDYAADDEEYEEKFRKETLAFFILATYGDGEPTDNAARFY 180

Qy 181 KWFVEGNDRGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVAEQGGKRIVPLVLGDDDQ 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 KWFVEGNDRGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVAEQGGKRIVPLVLGDDDQ 240

Qy 241 CIEDDFAAWRENVWPELDNLLRDEDDTTVSTTYTAAIPEYRVVFPDKSDSLISEANGHAN 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 CIEDDFAAWRENVWPELDNLLRDEDDTTVSTTYTAAIPEYRVVFPDKSDSLISEANGHAN 300

Qy 301 GYANGNTVYDAQHPCRSNVAVRKELHTPASDRSCTHLDFDIAGTGLSYGTGDHVGVYCDN 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 GYANGNTVYDAQHPCRSNVAVRKELHTPASDRSCTHLDFDIAGTGLSYGTGDHVGVYCDN 360

Qy 361 LSETVEEAERLLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCTLRTALTRYADLLNTP 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LSETVEEAERLLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCTLRTALTRYADLLNTP 420

Qy 421 KKSALLALAAYASDPNEADRLKYLASPAGKDEYAQSLVANQRSLLEVMAEFPSAKPPLGV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 KKSALLALAAYASDPNEADRLKYLASPAGKDEYAQSLVANQRSLLEVMAEFPSAKPPLGV 480

Qy 481 FFAAIA PRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGRIHKGVCSTWMKNAIPLEE 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 FFAAIA PRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGRIHKGVCSTWMKNAIPLEE 540

Qy 541 SRDCSWAPIFVRQSNFKLPADPKVPVIMIGPGTGLAPFRGFLQERLALKEEGAELGTAVF 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 SRDCSWAPIFVRQSNFKLPADPKVPVIMIGPGTGLAPFRGFLQERLALKEEGAELGTAVF 600

Qy 601 FFGCRNRKMDYIYEDELNHFLEIGALSELLVAFSREGPTKQYVQHKMAEKASDIWRMISD 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 FFGCRNRKMDYIYEDELNHFLEIGALSELLVAFSREGPTKQYVQHKMAEKASDIWRMISD 660

Qy 661 GAYVYVCGDAKGMARDVHRTLHTIAQEQGSMDSTQAEGFVKNLQMTGRYLRDVW 714
||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 GAYVYVCGDAKGMARDVHRTLHTIAQEQGSMDSTQAEGFVKNLQMTGRYLRDVW 714

Sequence alignment of Bassica napus F3’H of SEQ ID NO:8 (“Qy”) and Bassica napus F3’H of Xu (“Db”)

A1XBC6_BRANA
ID A1XBC6_BRANA Unreviewed; 511 AA.
AC A1XBC6;
DT 06-FEB-2007, integrated into UniProtKB/TrEMBL.
DT 06-FEB-2007, sequence version 1.
DT 22-FEB-2023, entry version 56.
DE SubName: Full=Flavonoid 3'-hydroxylase {ECO:0000313|EMBL:ABC58722.1};
OS Brassica napus (Rape).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Brassicaceae; Brassiceae; Brassica.
OX NCBI_TaxID=3708 {ECO:0000313|EMBL:ABC58722.1};
RN [1] {ECO:0000313|EMBL:ABC58722.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=5B {ECO:0000313|EMBL:ABC58722.1};
RA Xu B.-B., Zhang X.-K., Wang R., Xie L.-L., Li J.-N., Chai Y.-R.;
RL Submitted (DEC-2005) to the EMBL/GenBank/DDBJ databases.
RN [2] {ECO:0000313|EMBL:ABC58722.1}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=5B {ECO:0000313|EMBL:ABC58722.1};
RX PubMed=16618519; DOI=10.1016/j.jplph.2006.03.001;
RA Xu B.B., Li J.N., Zhang X.K., Wang R., Xie L.L., Chai Y.R.;
RT "Cloning and molecular characterization of a functional flavonoid 3'-
RT hydroxylase gene from Brassica napus.";
RL J. Plant Physiol. 164:350-363(2007).
CC -!- SIMILARITY: Belongs to the cytochrome P450 family.
CC {ECO:0000256|RuleBase:RU000461}.
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DR EMBL; DQ324378; ABC58722.1; -; Genomic_DNA.
DR EMBL; DQ324379; ABC58723.1; -; mRNA.
DR RefSeq; NP_001302883.1; NM_001315954.1.
DR GeneID; 106419056; -.
DR KEGG; bna:106419056; -.
DR OrthoDB; 2900138at2759; -.
DR BRENDA; 1.14.14.82; 944.
DR GO; GO:0016020; C:membrane; IEA:UniProtKB-KW.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR GO; GO:0004497; F:monooxygenase activity; IEA:UniProtKB-KW.
DR GO; GO:0016705; F:oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen; IEA:InterPro.
DR Gene3D; 1.10.630.10; Cytochrome P450; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR002401; Cyt_P450_E_grp-I.
DR InterPro; IPR036396; Cyt_P450_sf.
DR PANTHER; PTHR47944; CYTOCHROME P450 98A9; 1.
DR PANTHER; PTHR47944:SF4; OS09G0441700 PROTEIN; 1.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00463; EP450I.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; Cytochrome P450; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 2: Evidence at transcript level;
KW Heme {ECO:0000256|RuleBase:RU000461}; Iron {ECO:0000256|RuleBase:RU000461};
KW Membrane {ECO:0000256|SAM:Phobius};
KW Metal-binding {ECO:0000256|RuleBase:RU000461};
KW Monooxygenase {ECO:0000256|RuleBase:RU000461};
KW Oxidoreductase {ECO:0000256|RuleBase:RU000461};
KW Transmembrane {ECO:0000256|SAM:Phobius};
KW Transmembrane helix {ECO:0000256|SAM:Phobius}.
FT TRANSMEM 6..22
FT /note="Helical"
FT /evidence="ECO:0000256|SAM:Phobius"
SQ SEQUENCE 511 AA; 56622 MW; 81EE9FFFAF4213F2 CRC64;

Query Match 100.0%; Score 2656; Length 511;
Best Local Similarity 100.0%;
Matches 511; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIGNLPHMGPKPHQTLAAMVTTY 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIGNLPHMGPKPHQTLAAMVTTY 60

Qy 61 GPILHLRLGFADVVVAASKSVAEQFLKVHDANFASRPPNSGAKHMAYNYQDLVFAPYGQR 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 GPILHLRLGFADVVVAASKSVAEQFLKVHDANFASRPPNSGAKHMAYNYQDLVFAPYGQR 120

Qy 121 WRMLRKISSVHLFSAKALEDFKHVRQEEVGTLMRELARANTKPVNLGQLVNMCVLNALGR 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 WRMLRKISSVHLFSAKALEDFKHVRQEEVGTLMRELARANTKPVNLGQLVNMCVLNALGR 180

Qy 181 EMIGRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFVPALDCLDLQGVAGKMKRLHKR 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 EMIGRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFVPALDCLDLQGVAGKMKRLHKR 240

Qy 241 FDAFLSSILEEHEAMKNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKALLLNMFTA 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 FDAFLSSILEEHEAMKNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKALLLNMFTA 300

Qy 301 GTDTSASTVDWAIA ELIRHPEIMRKAQEELDSVVGRGRPINESDLSQLPYLQAVIKENFR 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 GTDTSASTVDWAIA ELIRHPEIMRKAQEELDSVVGRGRPINESDLSQLPYLQAVIKENFR 360

Qy 361 LHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIA RDPDQWSDPLTFRPERFLPGGE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIA RDPDQWSDPLTFRPERFLPGGE 420

Qy 421 KAGVDVKGNDFELIPFGAGRRICAGLSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNME 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 KAGVDVKGNDFELIPFGAGRRICAGLSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNME 480

Qy 481 ETYGITLQRAVPLVVHPKLRLDMSAYGLGSA 511
|||||||||||||||||||||||||||||||
Db 481 ETYGITLQRAVPLVVHPKLRLDMSAYGLGSA 511

Sequence alignment of cytochrome b5 of SEQ ID NO:98 (“Qy”) and cytochrome b5 of de Vetten (“Db”)

Q9ZSP7_PETHY
ID Q9ZSP7_PETHY Unreviewed; 149 AA.
AC Q9ZSP7;
DT 01-MAY-1999, integrated into UniProtKB/TrEMBL.
DT 01-MAY-1999, sequence version 1.
DT 24-JAN-2024, entry version 94.
DE SubName: Full=Cytochrome B5 {ECO:0000313|EMBL:AAD10774.1, ECO:0000313|EMBL:AAR89457.1};
GN Name=difF {ECO:0000313|EMBL:AAD10774.1};
OS Petunia hybrida (Petunia).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC asterids; lamiids; Solanales; Solanaceae; Petunioideae; Petunia.
OX NCBI_TaxID=4102 {ECO:0000313|EMBL:AAD10774.1};
RN [1] {ECO:0000313|EMBL:AAD10774.1}
RP NUCLEOTIDE SEQUENCE.
RX PubMed=9892710; DOI=10.1073/pnas.96.2.778;
RA de Vetten N., ter Horst J., van Schaik H.P., de Boer A., Mol J., Koes R.;
RT "A cytochrome b5 is required for full activity of flavonoid 3', 5'-
RT hydroxylase, a cytochrome P450 involved in the formation of blue flower
RT colors.";
RL Proc. Natl. Acad. Sci. U.S.A. 96:778-783(1999).
RN [2] {ECO:0000313|EMBL:AAR89457.1}
RP NUCLEOTIDE SEQUENCE.
RC TISSUE=Petal {ECO:0000313|EMBL:AAR89457.1};
RA Xiao X.G., Geng A.Q.;
RT "Molecular cloning of pigment-related cDNA encoding cytochrome B5 from
RT Petunia hybrida.";
RL Submitted (DEC-2003) to the EMBL/GenBank/DDBJ databases.
RN [3] {ECO:0000313|EMBL:ABC74800.1}
RP NUCLEOTIDE SEQUENCE.
RC TISSUE=Petal {ECO:0000313|EMBL:ABC74800.1};
RA Geng A.Q., Xiao X.G.;
RT "Molecular cloning of cytochrome B5 genomic DNA.";
RL Submitted (JAN-2006) to the EMBL/GenBank/DDBJ databases.
CC -!- SIMILARITY: Belongs to the cytochrome b5 family.
CC {ECO:0000256|ARBA:ARBA00038168, ECO:0000256|RuleBase:RU362121}.
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DR EMBL; AF098510; AAD10774.1; -; mRNA.
DR EMBL; AY502948; AAR89457.1; -; mRNA.
DR EMBL; DQ352143; ABC74800.1; -; Genomic_DNA.
DR AlphaFoldDB; Q9ZSP7; -.
DR SMR; Q9ZSP7; -.
DR GO; GO:0016020; C:membrane; IEA:UniProtKB-KW.
DR GO; GO:0020037; F:heme binding; IEA:UniProtKB-UniRule.
DR GO; GO:0046872; F:metal ion binding; IEA:UniProtKB-UniRule.
DR Gene3D; 3.10.120.10; Cytochrome b5-like heme/steroid binding domain; 1.
DR InterPro; IPR001199; Cyt_B5-like_heme/steroid-bd.
DR InterPro; IPR036400; Cyt_B5-like_heme/steroid_sf.
DR InterPro; IPR018506; Cyt_B5_heme-BS.
DR PANTHER; PTHR19359; CYTOCHROME B5; 1.
DR PANTHER; PTHR19359:SF14; CYTOCHROME B5-RELATED; 1.
DR Pfam; PF00173; Cyt-b5; 1.
DR PRINTS; PR00363; CYTOCHROMEB5.
DR SMART; SM01117; Cyt-b5; 1.
DR SUPFAM; SSF55856; Cytochrome b5-like heme/steroid binding domain; 1.
DR PROSITE; PS00191; CYTOCHROME_B5_1; 1.
DR PROSITE; PS50255; CYTOCHROME_B5_2; 1.
PE 2: Evidence at transcript level;
KW Heme {ECO:0000256|ARBA:ARBA00022617, ECO:0000256|RuleBase:RU362121};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|RuleBase:RU362121};
KW Membrane {ECO:0000256|RuleBase:RU362121};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723,
KW ECO:0000256|RuleBase:RU362121};
KW Transmembrane {ECO:0000256|RuleBase:RU362121};
KW Transmembrane helix {ECO:0000256|RuleBase:RU362121};
KW Transport {ECO:0000256|ARBA:ARBA00022448}.
FT TRANSMEM 121..140
FT /note="Helical"
FT /evidence="ECO:0000256|RuleBase:RU362121"
FT DOMAIN 4..80
FT /note="Cytochrome b5 heme-binding"
FT /evidence="ECO:0000259|PROSITE:PS50255"
SQ SEQUENCE 149 AA; 17125 MW; 0B22A4A733E18445 CRC64;

Query Match 100.0%; Score 771; Length 149;
Best Local Similarity 100.0%;
Matches 149; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLEEHPGGEEVLIESAGKDATKEFQD 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLEEHPGGEEVLIESAGKDATKEFQD 60

Qy 61 IGHSKAAKNLLFKYQIGYLQGYKASDDSELELNLVTDSIKEPNKAKEMKAYVIKEDPKPK 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 IGHSKAAKNLLFKYQIGYLQGYKASDDSELELNLVTDSIKEPNKAKEMKAYVIKEDPKPK 120

Qy 121 YLTFVEYLLPFLAAAFYLYYRYLTGALQF 149
|||||||||||||||||||||||||||||
Db 121 YLTFVEYLLPFLAAAFYLYYRYLTGALQF 149

Sequence alignment of F3’H of SEQ ID NO:49 (“Qy”) and F3’H of A0A1Q3CVI5 (“Db”)

A0A1Q3CVI5_CEPFO
ID A0A1Q3CVI5_CEPFO Unreviewed; 511 AA.
AC A0A1Q3CVI5;
DT 12-APR-2017, integrated into UniProtKB/TrEMBL.
DT 12-APR-2017, sequence version 1.
DT 28-JAN-2026, entry version 24.
DE RecName: Full=Flavonoid 3'-monooxygenase {ECO:0000256|ARBA:ARBA00070032};
DE EC=1.14.14.82 {ECO:0000256|ARBA:ARBA00066562};
DE AltName: Full=Flavonoid 3'-hydroxylase {ECO:0000256|ARBA:ARBA00081480};
GN ORFNames=CFOL_v3_27507 {ECO:0000313|EMBL:GAV84063.1};
OS Cephalotus follicularis (Albany pitcher plant).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; fabids; Oxalidales; Cephalotaceae; Cephalotus.
OX NCBI_TaxID=3775 {ECO:0000313|EMBL:GAV84063.1, ECO:0000313|Proteomes:UP000187406};
RN [1] {ECO:0000313|Proteomes:UP000187406}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. St1 {ECO:0000313|Proteomes:UP000187406};
RA Fukushima K., Hasebe M., Fang X.;
RT "Cephalotus genome sequencing.";
RL Submitted (APR-2016) to the EMBL/GenBank/DDBJ databases.
CC -!- FUNCTION: Catalyzes the 3'-hydroxylation of the flavonoid B-ring to the
CC 3',4'-hydroxylated state. Convert naringenin to eriodictyol and
CC dihydrokaempferol to dihydroquercetin. {ECO:0000256|ARBA:ARBA00055665}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a 3'-unsubstituted flavone + reduced [NADPH--hemoprotein
CC reductase] + O2 = a 3'-hydroxyflavone + oxidized [NADPH--hemoprotein
CC reductase] + H2O + H(+); Xref=Rhea:RHEA:16337, Rhea:RHEA-COMP:11964,
CC Rhea:RHEA-COMP:11965, ChEBI:CHEBI:15377, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:15379, ChEBI:CHEBI:27741, ChEBI:CHEBI:57618,
CC ChEBI:CHEBI:58210, ChEBI:CHEBI:138726; EC=1.14.14.82;
CC Evidence={ECO:0000256|ARBA:ARBA00052910};
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413;
CC Evidence={ECO:0000256|ARBA:ARBA00001971,
CC ECO:0000256|PIRSR:PIRSR602401-1};
CC -!- PATHWAY: Secondary metabolite biosynthesis; flavonoid biosynthesis.
CC {ECO:0000256|ARBA:ARBA00004966}.
CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane
CC {ECO:0000256|ARBA:ARBA00004643}; Single-pass type III membrane protein
CC {ECO:0000256|ARBA:ARBA00004643}.
CC -!- SIMILARITY: Belongs to the cytochrome P450 family.
CC {ECO:0000256|ARBA:ARBA00010617, ECO:0000256|RuleBase:RU000461}.
CC -!- CAUTION: The sequence shown here is derived from an EMBL/GenBank/DDBJ
CC whole genome shotgun (WGS) entry which is preliminary data.
CC {ECO:0000313|EMBL:GAV84063.1}.
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CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; BDDD01003101; GAV84063.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A1Q3CVI5; -.
DR FunCoup; A0A1Q3CVI5; 335.
DR STRING; 3775.A0A1Q3CVI5; -.
DR InParanoid; A0A1Q3CVI5; -.
DR OrthoDB; 2789670at2759; -.
DR Proteomes; UP000187406; Unassembled WGS sequence.
DR GO; GO:0005789; C:endoplasmic reticulum membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0016711; F:flavonoid 3'-monooxygenase activity; IEA:UniProtKB-EC.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR GO; GO:0009813; P:flavonoid biosynthetic process; IEA:UniProtKB-KW.
DR FunFam; 1.10.630.10:FF:000056; Red aleurone1; 1.
DR Gene3D; 1.10.630.10; Cytochrome P450; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR002401; Cyt_P450_E_grp-I.
DR InterPro; IPR036396; Cyt_P450_sf.
DR PANTHER; PTHR47944; CYTOCHROME P450 98A9; 1.
DR PANTHER; PTHR47944:SF18; FLAVONOID 3'-MONOOXYGENASE; 1.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00463; EP450I.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; Cytochrome P450; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 3: Inferred from homology;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW Heme {ECO:0000256|ARBA:ARBA00022617, ECO:0000256|PIRSR:PIRSR602401-1};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|PIRSR:PIRSR602401-1};
KW Membrane {ECO:0000256|ARBA:ARBA00023136};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723,
KW ECO:0000256|PIRSR:PIRSR602401-1};
KW Monooxygenase {ECO:0000256|ARBA:ARBA00023033,
KW ECO:0000256|RuleBase:RU000461}; NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000256|RuleBase:RU000461};
KW Reference proteome {ECO:0000313|Proteomes:UP000187406};
KW Transmembrane {ECO:0000256|ARBA:ARBA00022692};
KW Transmembrane helix {ECO:0000256|ARBA:ARBA00022989}.
FT BINDING 448
FT /ligand="heme"
FT /ligand_id="ChEBI:CHEBI:30413"
FT /ligand_part="Fe"
FT /ligand_part_id="ChEBI:CHEBI:18248"
FT /note="axial binding residue"
FT /evidence="ECO:0000256|PIRSR:PIRSR602401-1"
SQ SEQUENCE 511 AA; 56768 MW; 4737C34CAADDB375 CRC64;

Query Match 100.0%; Score 2644; Length 511;
Best Local Similarity 100.0%;
Matches 511; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIGNLPHMGQMPHHSLAAMARTY 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIGNLPHMGQMPHHSLAAMARTY 60

Qy 61 GPLMHLRLGFVDVIVAASASVASQLLKTHDANFSSRPHNSGAKYIAYNYQDLVFAPYGPR 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 GPLMHLRLGFVDVIVAASASVASQLLKTHDANFSSRPHNSGAKYIAYNYQDLVFAPYGPR 120

Qy 121 WRMLRKISSVHLFSGKALDDYRHVRQEEVAVLIRALARAESKQAVNLGQLLNVCTANALG 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 WRMLRKISSVHLFSGKALDDYRHVRQEEVAVLIRALARAESKQAVNLGQLLNVCTANALG 180

Qy 181 RVMLGRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIGDFIPALDWLDLQGVAAKMKN 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 RVMLGRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIGDFIPALDWLDLQGVAAKMKN 240

Qy 241 LHKRFDTFLTGLLEEHKKMLVGDGGSEKHKDLLSTLISLKDSADDEGLKLTDTEIKALLL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LHKRFDTFLTGLLEEHKKMLVGDGGSEKHKDLLSTLISLKDSADDEGLKLTDTEIKALLL 300

Qy 301 NMFTAGTDTSSSTVEWAIA ELIRHPKILAQVLKELDTVVGRDRLVTDLDLPQLTYLQAVI 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 NMFTAGTDTSSSTVEWAIA ELIRHPKILAQVLKELDTVVGRDRLVTDLDLPQLTYLQAVI 360

Qy 361 KETFRLHPSTPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIA RDPKEWAEPLEFRPERF 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 KETFRLHPSTPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIA RDPKEWAEPLEFRPERF 420

Qy 421 LPGGEKPNVDIKGNDFEVIPFGAGRRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPE 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 LPGGEKPNVDIKGNDFEVIPFGAGRRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPE 480

Qy 481 DLSMEEAYGLTLQRAEPLMVHPRPRLSPNVY 511
|||||||||||||||||||||||||||||||
Db 481 DLSMEEAYGLTLQRAEPLMVHPRPRLSPNVY 511

Sequence alignment of F3’H of SEQ ID NO:50 (“Qy”) and F3’H of A0A061G4T4 (“Db”)

A0A061G4T4_THECC
ID A0A061G4T4_THECC Unreviewed; 507 AA.
AC A0A061G4T4;
DT 03-SEP-2014, integrated into UniProtKB/TrEMBL.
DT 03-SEP-2014, sequence version 1.
DT 28-JAN-2026, entry version 62.
DE RecName: Full=Flavonoid 3'-monooxygenase {ECO:0000256|ARBA:ARBA00070032};
DE EC=1.14.14.82 {ECO:0000256|ARBA:ARBA00066562};
DE AltName: Full=Flavonoid 3'-hydroxylase {ECO:0000256|ARBA:ARBA00081480};
GN ORFNames=TCM_014241 {ECO:0000313|EMBL:EOY22049.1};
OS Theobroma cacao (Cacao) (Cocoa).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Malvales; Malvaceae; Byttnerioideae; Theobroma.
OX NCBI_TaxID=3641 {ECO:0000313|EMBL:EOY22049.1, ECO:0000313|Proteomes:UP000026915};
RN [1] {ECO:0000313|EMBL:EOY22049.1, ECO:0000313|Proteomes:UP000026915}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Matina 1-6 {ECO:0000313|Proteomes:UP000026915};
RX PubMed=23731509; DOI=10.1186/gb-2013-14-6-r53;
RA Motamayor J.C., Mockaitis K., Schmutz J., Haiminen N., Iii D.L.,
RA Cornejo O., Findley S.D., Zheng P., Utro F., Royaert S., Saski C.,
RA Jenkins J., Podicheti R., Zhao M., Scheffler B.E., Stack J.C., Feltus F.A.,
RA Mustiga G.M., Amores F., Phillips W., Marelli J.P., May G.D., Shapiro H.,
RA Ma J., Bustamante C.D., Schnell R.J., Main D., Gilbert D., Parida L.,
RA Kuhn D.N.;
RT "The genome sequence of the most widely cultivated cacao type and its use
RT to identify candidate genes regulating pod color.";
RL Genome Biol. 14:R53.1-R53.24(2013).
CC -!- FUNCTION: Catalyzes the 3'-hydroxylation of the flavonoid B-ring to the
CC 3',4'-hydroxylated state. Convert naringenin to eriodictyol and
CC dihydrokaempferol to dihydroquercetin. {ECO:0000256|ARBA:ARBA00055665}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a 3'-unsubstituted flavone + reduced [NADPH--hemoprotein
CC reductase] + O2 = a 3'-hydroxyflavone + oxidized [NADPH--hemoprotein
CC reductase] + H2O + H(+); Xref=Rhea:RHEA:16337, Rhea:RHEA-COMP:11964,
CC Rhea:RHEA-COMP:11965, ChEBI:CHEBI:15377, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:15379, ChEBI:CHEBI:27741, ChEBI:CHEBI:57618,
CC ChEBI:CHEBI:58210, ChEBI:CHEBI:138726; EC=1.14.14.82;
CC Evidence={ECO:0000256|ARBA:ARBA00052910};
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413;
CC Evidence={ECO:0000256|ARBA:ARBA00001971,
CC ECO:0000256|PIRSR:PIRSR602401-1};
CC -!- PATHWAY: Secondary metabolite biosynthesis; flavonoid biosynthesis.
CC {ECO:0000256|ARBA:ARBA00004966}.
CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane
CC {ECO:0000256|ARBA:ARBA00004643}; Single-pass type III membrane protein
CC {ECO:0000256|ARBA:ARBA00004643}.
CC -!- SIMILARITY: Belongs to the cytochrome P450 family.
CC {ECO:0000256|ARBA:ARBA00010617, ECO:0000256|RuleBase:RU000461}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; CM001881; EOY22049.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A061G4T4; -.
DR SMR; A0A061G4T4; -.
DR FunCoup; A0A061G4T4; 349.
DR STRING; 3641.A0A061G4T4; -.
DR Gramene; EOY22049; EOY22049; TCM_014241.
DR Gramene; Tc03v2_t008270.1; Tc03v2_p008270.1; Tc03v2_g008270.
DR KEGG; tcc:18604820; -.
DR eggNOG; KOG0156; Eukaryota.
DR InParanoid; A0A061G4T4; -.
DR OMA; QIRLGNC; -.
DR OrthoDB; 2789670at2759; -.
DR Proteomes; UP000026915; Chromosome 3.
DR GO; GO:0005789; C:endoplasmic reticulum membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0016020; C:membrane; IBA:GO_Central.
DR GO; GO:0016711; F:flavonoid 3'-monooxygenase activity; IEA:UniProtKB-EC.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR GO; GO:0016709; F:oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen, NAD(P)H as one donor, and incorporation of one atom of oxygen; IBA:GO_Central.
DR GO; GO:0009813; P:flavonoid biosynthetic process; IEA:UniProtKB-KW.
DR FunFam; 1.10.630.10:FF:000056; Red aleurone1; 1.
DR Gene3D; 1.10.630.10; Cytochrome P450; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR002401; Cyt_P450_E_grp-I.
DR InterPro; IPR036396; Cyt_P450_sf.
DR PANTHER; PTHR47944; CYTOCHROME P450 98A9; 1.
DR PANTHER; PTHR47944:SF18; FLAVONOID 3'-MONOOXYGENASE; 1.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00463; EP450I.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; Cytochrome P450; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 3: Inferred from homology;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW Heme {ECO:0000256|ARBA:ARBA00022617, ECO:0000256|PIRSR:PIRSR602401-1};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|PIRSR:PIRSR602401-1};
KW Membrane {ECO:0000256|ARBA:ARBA00023136, ECO:0000256|SAM:Phobius};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723,
KW ECO:0000256|PIRSR:PIRSR602401-1};
KW Monooxygenase {ECO:0000256|ARBA:ARBA00023033,
KW ECO:0000256|RuleBase:RU000461}; NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000256|RuleBase:RU000461};
KW Reference proteome {ECO:0000313|Proteomes:UP000026915};
KW Transmembrane {ECO:0000256|ARBA:ARBA00022692, ECO:0000256|SAM:Phobius};
KW Transmembrane helix {ECO:0000256|ARBA:ARBA00022989,
KW ECO:0000256|SAM:Phobius}.
FT TRANSMEM 6..22
FT /note="Helical"
FT /evidence="ECO:0000256|SAM:Phobius"
FT BINDING 444
FT /ligand="heme"
FT /ligand_id="ChEBI:CHEBI:30413"
FT /ligand_part="Fe"
FT /ligand_part_id="ChEBI:CHEBI:18248"
FT /note="axial binding residue"
FT /evidence="ECO:0000256|PIRSR:PIRSR602401-1"
SQ SEQUENCE 507 AA; 56258 MW; E870BCAB1D4F8344 CRC64;

Query Match 100.0%; Score 2625; Length 507;
Best Local Similarity 100.0%;
Matches 507; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPIIGNLPHMGPVPHHSLAALAKV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPIIGNLPHMGPVPHHSLAALAKV 60

Qy 61 YGPLMHLRLGFVDVVVAASASVAAQFLKVHDANFSSRPPNSGAKYVAYNYQDLVFAPYGP 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 YGPLMHLRLGFVDVVVAASASVAAQFLKVHDANFSSRPPNSGAKYVAYNYQDLVFAPYGP 120

Qy 121 RWRMLRKISSVHLFSGKALDDFRHVRQDEVGVLVRALADAKTKVNLGQLLNVCTVNALGR 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 RWRMLRKISSVHLFSGKALDDFRHVRQDEVGVLVRALADAKTKVNLGQLLNVCTVNALGR 180

Qy 181 VMLGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNIGDFIPALEWLDLQGVQAKMKKL 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 VMLGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNIGDFIPALEWLDLQGVQAKMKKL 240

Qy 241 HKRFDRFLSAILEEHKIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKALLLNMFT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 HKRFDRFLSAILEEHKIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKALLLNMFT 300

Qy 301 AGTDTSSSTVEWAIA ELIRHPKILAQVRKELDSVVGRDRLVSDLDLPNLTYFQAVIKETF 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 AGTDTSSSTVEWAIA ELIRHPKILAQVRKELDSVVGRDRLVSDLDLPNLTYFQAVIKETF 360

Qy 361 RLHPSTPLSLPRMASESCEINGYHIPKGATLLVNVWAIA RDPDEWKDPLEFRPERFLPGG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 RLHPSTPLSLPRMASESCEINGYHIPKGATLLVNVWAIA RDPDEWKDPLEFRPERFLPGG 420

Qy 421 ERPNADVRGNDFEVIPFGAGRRICAGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLNM 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ERPNADVRGNDFEVIPFGAGRRICAGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLNM 480

Qy 481 EEAFGLTLQRAAPLMVHPRPRLSPRAY 507
|||||||||||||||||||||||||||
Db 481 EEAFGLTLQRAAPLMVHPRPRLSPRAY 507

Sequence alignment of F3’H of SEQ ID NO:51 (“Qy”) and F3’H of Q38L00 (“Db”)

Q38L00_GERHY
ID Q38L00_GERHY Unreviewed; 512 AA.
AC Q38L00;
DT 22-NOV-2005, integrated into UniProtKB/TrEMBL.
DT 22-NOV-2005, sequence version 1.
DT 28-JAN-2026, entry version 68.
DE RecName: Full=Flavonoid 3'-monooxygenase {ECO:0000256|ARBA:ARBA00070032};
DE EC=1.14.14.82 {ECO:0000256|ARBA:ARBA00066562};
DE AltName: Full=Flavonoid 3'-hydroxylase {ECO:0000256|ARBA:ARBA00081480};
OS Gerbera hybrida (Daisy).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC asterids; campanulids; Asterales; Asteraceae; Mutisioideae; Mutisieae;
OC Gerbera.
OX NCBI_TaxID=18101 {ECO:0000313|EMBL:ABA64468.1};
RN [1] {ECO:0000313|EMBL:ABA64468.1}
RP NUCLEOTIDE SEQUENCE.
RA Seitz C., Eder C., Deiml B., Kellner S., Martens S., Forkmann G.;
RT "Cloning, Functional Identification and Sequence Analysis of F3'H and
RT F3'5'H cDNAs Reveals Independent Evolution of F3'5'H in the Asteraceae
RT Family.";
RL Submitted (SEP-2005) to the EMBL/GenBank/DDBJ databases.
CC -!- FUNCTION: Catalyzes the 3'-hydroxylation of the flavonoid B-ring to the
CC 3',4'-hydroxylated state. Convert naringenin to eriodictyol and
CC dihydrokaempferol to dihydroquercetin. {ECO:0000256|ARBA:ARBA00055665}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a 3'-unsubstituted flavone + reduced [NADPH--hemoprotein
CC reductase] + O2 = a 3'-hydroxyflavone + oxidized [NADPH--hemoprotein
CC reductase] + H2O + H(+); Xref=Rhea:RHEA:16337, Rhea:RHEA-COMP:11964,
CC Rhea:RHEA-COMP:11965, ChEBI:CHEBI:15377, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:15379, ChEBI:CHEBI:27741, ChEBI:CHEBI:57618,
CC ChEBI:CHEBI:58210, ChEBI:CHEBI:138726; EC=1.14.14.82;
CC Evidence={ECO:0000256|ARBA:ARBA00052910};
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413;
CC Evidence={ECO:0000256|ARBA:ARBA00001971,
CC ECO:0000256|PIRSR:PIRSR602401-1};
CC -!- PATHWAY: Secondary metabolite biosynthesis; flavonoid biosynthesis.
CC {ECO:0000256|ARBA:ARBA00004966}.
CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane
CC {ECO:0000256|ARBA:ARBA00004643}; Single-pass type III membrane protein
CC {ECO:0000256|ARBA:ARBA00004643}.
CC -!- SIMILARITY: Belongs to the cytochrome P450 family.
CC {ECO:0000256|ARBA:ARBA00010617, ECO:0000256|RuleBase:RU000461}.
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DR EMBL; DQ218417; ABA64468.1; -; mRNA.
DR AlphaFoldDB; Q38L00; -.
DR SMR; Q38L00; -.
DR BRENDA; 1.14.14.82; 2420.
DR GO; GO:0005789; C:endoplasmic reticulum membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0016711; F:flavonoid 3'-monooxygenase activity; IEA:UniProtKB-EC.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR GO; GO:0009813; P:flavonoid biosynthetic process; IEA:UniProtKB-KW.
DR FunFam; 1.10.630.10:FF:000056; Red aleurone1; 1.
DR Gene3D; 1.10.630.10; Cytochrome P450; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR002401; Cyt_P450_E_grp-I.
DR InterPro; IPR036396; Cyt_P450_sf.
DR PANTHER; PTHR47944; CYTOCHROME P450 98A9; 1.
DR PANTHER; PTHR47944:SF18; FLAVONOID 3'-MONOOXYGENASE; 1.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00463; EP450I.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; Cytochrome P450; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 2: Evidence at transcript level;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW Heme {ECO:0000256|ARBA:ARBA00022617, ECO:0000256|PIRSR:PIRSR602401-1};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|PIRSR:PIRSR602401-1};
KW Membrane {ECO:0000256|ARBA:ARBA00023136};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723,
KW ECO:0000256|PIRSR:PIRSR602401-1};
KW Monooxygenase {ECO:0000256|ARBA:ARBA00023033,
KW ECO:0000256|RuleBase:RU000461}; NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000256|RuleBase:RU000461};
KW Transmembrane {ECO:0000256|ARBA:ARBA00022692};
KW Transmembrane helix {ECO:0000256|ARBA:ARBA00022989}.
FT BINDING 444
FT /ligand="heme"
FT /ligand_id="ChEBI:CHEBI:30413"
FT /ligand_part="Fe"
FT /ligand_part_id="ChEBI:CHEBI:18248"
FT /note="axial binding residue"
FT /evidence="ECO:0000256|PIRSR:PIRSR602401-1"
SQ SEQUENCE 512 AA; 56318 MW; 7CAF4D140111DDE2 CRC64;

Query Match 100.0%; Score 2661; Length 512;
Best Local Similarity 100.0%;
Matches 512; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPWPVVGNLPHLGTIPHHSLAAMAK 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPWPVVGNLPHLGTIPHHSLAAMAK 60

Qy 61 KYGPLMHLRLGFVDVVVAASASVAAQFLKTHDANFADRPPNSGAKHIAYNYQDLVFAPYG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 KYGPLMHLRLGFVDVVVAASASVAAQFLKTHDANFADRPPNSGAKHIAYNYQDLVFAPYG 120

Qy 121 PRWRMLRKICSVHLFSTKALDDFRHVRQEEVAILARALVGAGKSPVKLGQLLNVCTTNAL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 PRWRMLRKICSVHLFSTKALDDFRHVRQEEVAILARALVGAGKSPVKLGQLLNVCTTNAL 180

Qy 181 ARVMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIGDFIPVLDWLDLQGVTKKMKKLHA 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ARVMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIGDFIPVLDWLDLQGVTKKMKKLHA 240

Qy 241 KFDSFLNTILEEHKTGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEIKALLLNLFT 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 KFDSFLNTILEEHKTGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEIKALLLNLFT 300

Qy 301 AGTDTSSSTIEWAIA ELIRNPQLLNQARKEMDTIVGQDRLVTESDLGQLTFLQAIIKETF 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 AGTDTSSSTIEWAIA ELIRNPQLLNQARKEMDTIVGQDRLVTESDLGQLTFLQAIIKETF 360

Qy 361 RLHPSTPLSLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIWADPLEFQPTRFLPGG 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 RLHPSTPLSLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIWADPLEFQPTRFLPGG 420

Qy 421 EKPNTDIKGNDFEVIPFGAGRRICVGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNM 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 EKPNTDIKGNDFEVIPFGAGRRICVGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNM 480

Qy 481 EEAYGLTLQRAAPLVVHPRPRLAPHVYETTKV 512
||||||||||||||||||||||||||||||||
Db 481 EEAYGLTLQRAAPLVVHPRPRLAPHVYETTKV 512

Sequence alignment of F3’H of SEQ ID NO:52 (“Qy”) and F3’H of XP_008791304.2 (“Db”)

A0A8B7C491_PHODC
ID A0A8B7C491_PHODC Unreviewed; 517 AA.
AC A0A8B7C491;
DT 19-JAN-2022, integrated into UniProtKB/TrEMBL.
DT 19-JAN-2022, sequence version 1.
DT 28-JAN-2026, entry version 21.
DE SubName: Full=Flavonoid 3'-monooxygenase CYP75B137-like {ECO:0000313|RefSeq:XP_008791304.2};
GN Name=LOC103708250 {ECO:0000313|RefSeq:XP_008791304.2};
OS Phoenix dactylifera (Date palm).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; Liliopsida; Arecaceae; Coryphoideae;
OC Phoeniceae; Phoenix.
OX NCBI_TaxID=42345 {ECO:0000313|Proteomes:UP000228380, ECO:0000313|RefSeq:XP_008791304.2};
RN [1] {ECO:0000313|Proteomes:UP000228380}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Khalas {ECO:0000313|Proteomes:UP000228380};
RX PubMed=31615981;
RA Hazzouri K.M., Gros-Balthazard M., Flowers J.M., Copetti D., Lemansour A.,
RA Lebrun M., Masmoudi K., Ferrand S., Dhar M.I., Fresquez Z.A., Rosas U.,
RA Zhang J., Talag J., Lee S., Kudrna D., Powell R.F., Leitch I.J.,
RA Krueger R.R., Wing R.A., Amiri K.M.A., Purugganan M.D.;
RT "Genome-wide association mapping of date palm fruit traits.";
RL Nat. Commun. 10:4680-4680(2019).
RN [2] {ECO:0000313|RefSeq:XP_008791304.2}
RP IDENTIFICATION.
RC TISSUE=Young leaves {ECO:0000313|RefSeq:XP_008791304.2};
RG RefSeq;
RL Submitted (AUG-2025) to UniProtKB.
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413;
CC Evidence={ECO:0000256|ARBA:ARBA00001971,
CC ECO:0000256|PIRSR:PIRSR602401-1};
CC -!- SIMILARITY: Belongs to the cytochrome P450 family.
CC {ECO:0000256|ARBA:ARBA00010617, ECO:0000256|RuleBase:RU000461}.
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DR RefSeq; XP_008791304.2; XM_008793082.4.
DR AlphaFoldDB; A0A8B7C491; -.
DR GeneID; 103708250; -.
DR KEGG; pda:103708250; -.
DR OrthoDB; 2789670at2759; -.
DR Proteomes; UP000228380; Chromosome 15.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR GO; GO:0004497; F:monooxygenase activity; IEA:UniProtKB-KW.
DR GO; GO:0016705; F:oxidoreductase activity, acting on paired donors, with incorporation or reduction of molecular oxygen; IEA:InterPro.
DR FunFam; 1.10.630.10:FF:000026; Cytochrome P450 82C4; 1.
DR Gene3D; 1.10.630.10; Cytochrome P450; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR002401; Cyt_P450_E_grp-I.
DR InterPro; IPR036396; Cyt_P450_sf.
DR PANTHER; PTHR47944; CYTOCHROME P450 98A9; 1.
DR PANTHER; PTHR47944:SF18; FLAVONOID 3'-MONOOXYGENASE; 1.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00463; EP450I.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; Cytochrome P450; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 3: Inferred from homology;
KW Heme {ECO:0000256|ARBA:ARBA00022617, ECO:0000256|PIRSR:PIRSR602401-1};
KW Iron {ECO:0000256|ARBA:ARBA00023004, ECO:0000256|PIRSR:PIRSR602401-1};
KW Membrane {ECO:0000256|SAM:Phobius};
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723,
KW ECO:0000256|PIRSR:PIRSR602401-1};
KW Monooxygenase {ECO:0000256|ARBA:ARBA00023033,
KW ECO:0000256|RuleBase:RU000461}; NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000256|RuleBase:RU000461};
KW Reference proteome {ECO:0000313|Proteomes:UP000228380};
KW Transmembrane {ECO:0000256|SAM:Phobius};
KW Transmembrane helix {ECO:0000256|SAM:Phobius}.
FT TRANSMEM 6..22
FT /note="Helical"
FT /evidence="ECO:0000256|SAM:Phobius"
FT BINDING 452
FT /ligand="heme"
FT /ligand_id="ChEBI:CHEBI:30413"
FT /ligand_part="Fe"
FT /ligand_part_id="ChEBI:CHEBI:18248"
FT /note="axial binding residue"
FT /evidence="ECO:0000256|PIRSR:PIRSR602401-1"
SQ SEQUENCE 517 AA; 56852 MW; 55FC2FAB8F5807F7 CRC64;

Query Match 100.0%; Score 2698; Length 517;
Best Local Similarity 100.0%;
Matches 517; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGPRGWPVLGNLPQLGPKPHHTLHA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGPRGWPVLGNLPQLGPKPHHTLHA 60

Qy 61 LSRAHGPLFRLRLGSVDVVVAASAAVAAQFLRAHDANFSNRPPNSGAEHIAYNYQDLVFA 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LSRAHGPLFRLRLGSVDVVVAASAAVAAQFLRAHDANFSNRPPNSGAEHIAYNYQDLVFA 120

Qy 121 PYGPGWRARRKLLNVHLFSGKALEDLRPVREGELALLVRALRDRAGANELVDLGRAANKC 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 PYGPGWRARRKLLNVHLFSGKALEDLRPVREGELALLVRALRDRAGANELVDLGRAANKC 180

Qy 181 ATNALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAGVFNVGDFVPGIGWLDLQGVVRR 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 ATNALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAGVFNVGDFVPGIGWLDLQGVVRR 240

Qy 241 MKELHRRYDGFLDGLIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGEGAKLTDTDIK 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 MKELHRRYDGFLDGLIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGEGAKLTDTDIK 300

Qy 301 ALLLNLFTAGTDTTSSTVEWALSELVKHPDILRKAQLELDSVVGGDRLVSESDLPNLPFM 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 ALLLNLFTAGTDTTSSTVEWALSELVKHPDILRKAQLELDSVVGGDRLVSESDLPNLPFM 360

Qy 361 QAIIKETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNVWAIA RDPAVWRDPLEFR 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 QAIIKETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNVWAIA RDPAVWRDPLEFR 420

Qy 421 PARFLPDGGCEGMDVKGNDFGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWDLPEG 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 PARFLPDGGCEGMDVKGNDFGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWDLPEG 480

Qy 481 QMPEKLDMEEAYGLTLQRATPLMVHPVPRLAPTAYQS 517
|||||||||||||||||||||||||||||||||||||
Db 481 QMPEKLDMEEAYGLTLQRATPLMVHPVPRLAPTAYQS 517

Sequence alignment of CPR of SEQ ID NO:53 (“Qy”) and CPR of XP_028084858.1 (“Db”)

Title: US-17-720-020-53
Perfect score: 5219
Sequence: 1 MASNSNLIRAIESALGVSFG..........KAEVIVKKLQMEGRYLRDVW 1007

Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5

Searched: 1 seqs, 1007 residues

Total number of hits satisfying chosen parameters: 1

Minimum DB seq length: 0
Maximum DB seq length: inf

Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 50 summaries

Database : XP_028084858.1.fasta:*

SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 5219 100.0 1007 1 XP_028084858.1 NADPH--cytochrome

ALIGNMENTS

RESULT 1
XP_028084858.1

Query Match 100.0%; Score 5219; DB 1; Length 1007;
Best Local Similarity 100.0%;
Matches 1007; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPV 60

Qy 61 VISKPLLVEEEEEEDEVEAGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEKAIVKVV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 VISKPLLVEEEEEEDEVEAGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEKAIVKVV 120

Qy 121 DLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY 180

Qy 181 GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELD 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELD 240

Qy 241 QLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSN 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 QLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSN 300

Qy 301 QQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 QQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF 360

Qy 361 LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEE 420

Qy 421 IKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTE 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTE 480

Qy 481 ENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 ENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD 540

Qy 541 FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNA 600

Qy 601 SYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEE 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 SYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEE 660

Qy 661 AANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 AANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA 720

Qy 721 LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAP 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAP 780

Qy 781 RLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSA 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 RLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSA 840

Qy 841 PIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 PIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR 900

Qy 901 RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVC 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVC 960

Qy 961 GDAKGMARDVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLRDVW 1007
|||||||||||||||||||||||||||||||||||||||||||||||
Db 961 GDAKGMARDVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLRDVW 1007

Sequence alignment of CPR of SEQ ID NO:54 (“Qy”) and CPR of XP_028084858.1 (“Db”)

Title: US-17-720-020-54
Perfect score: 5219
Sequence: 1 MASNSNLIRAIESALGVSFG..........KAEVIVKKLQMEGRYLRDVW 1007

Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5

Searched: 1 seqs, 1007 residues

Total number of hits satisfying chosen parameters: 1

Minimum DB seq length: 0
Maximum DB seq length: inf

Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 50 summaries

Database : XP_028084858.1.fasta:*

SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 5219 100.0 1007 1 XP_028084858.1 NADPH--cytochrome

ALIGNMENTS

RESULT 1
XP_028084858.1

Query Match 100.0%; Score 5219; DB 1; Length 1007;
Best Local Similarity 100.0%;
Matches 1007; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPV 60

Qy 61 VISKPLLVEEEEEEDEVEAGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEKAIVKVV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 VISKPLLVEEEEEEDEVEAGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEKAIVKVV 120

Qy 121 DLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY 180

Qy 181 GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELD 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELD 240

Qy 241 QLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSN 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 QLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSN 300

Qy 301 QQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 QQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF 360

Qy 361 LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEE 420

Qy 421 IKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTE 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTE 480

Qy 481 ENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 ENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD 540

Qy 541 FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNA 600

Qy 601 SYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEE 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 SYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEE 660

Qy 661 AANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 AANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA 720

Qy 721 LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAP 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAP 780

Qy 781 RLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSA 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 RLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSA 840

Qy 841 PIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 PIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR 900

Qy 901 RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVC 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVC 960

Qy 961 GDAKGMARDVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLRDVW 1007
|||||||||||||||||||||||||||||||||||||||||||||||
Db 961 GDAKGMARDVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLRDVW 1007

Sequence alignment of CPR of SEQ ID NO:55 (“Qy”) and CPR of A0A0D3DGY (“Db”)

A0A0D3DGY4_BRAOL
ID A0A0D3DGY4_BRAOL Unreviewed; 708 AA.
AC A0A0D3DGY4;
DT 29-APR-2015, integrated into UniProtKB/TrEMBL.
DT 29-APR-2015, sequence version 1.
DT 08-OCT-2025, entry version 53.
DE RecName: Full=NADPH--cytochrome P450 reductase {ECO:0000256|HAMAP-Rule:MF_03212, ECO:0000256|PIRNR:PIRNR000208};
DE Short=CPR {ECO:0000256|HAMAP-Rule:MF_03212};
DE Short=P450R {ECO:0000256|HAMAP-Rule:MF_03212};
DE EC=1.6.2.4 {ECO:0000256|HAMAP-Rule:MF_03212, ECO:0000256|PIRNR:PIRNR000208};
OS Brassica oleracea var. oleracea.
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Brassicales; Brassicaceae; Brassiceae; Brassica.
OX NCBI_TaxID=109376 {ECO:0000313|EnsemblPlants:Bo7g114320.1, ECO:0000313|Proteomes:UP000032141};
RN [1] {ECO:0000313|EnsemblPlants:Bo7g114320.1, ECO:0000313|Proteomes:UP000032141}
RP NUCLEOTIDE SEQUENCE.
RC STRAIN=cv. TO1000 {ECO:0000313|EnsemblPlants:Bo7g114320.1,
RC ECO:0000313|Proteomes:UP000032141};
RX PubMed=24916971; DOI=10.1186/gb-2014-15-6-r77;
RA Parkin I.A., Koh C., Tang H., Robinson S.J., Kagale S., Clarke W.E.,
RA Town C.D., Nixon J., Krishnakumar V., Bidwell S.L., Denoeud F., Belcram H.,
RA Links M.G., Just J., Clarke C., Bender T., Huebert T., Mason A.S.,
RA Pires J.C., Barker G., Moore J., Walley P.G., Manoli S., Batley J.,
RA Edwards D., Nelson M.N., Wang X., Paterson A.H., King G., Bancroft I.,
RA Chalhoub B., Sharpe A.G.;
RT "Transcriptome and methylome profiling reveals relics of genome dominance
RT in the mesopolyploid Brassica oleracea.";
RL Genome Biol. 15:R77-R77(2014).
RN [2] {ECO:0000313|EnsemblPlants:Bo7g114320.1}
RP IDENTIFICATION.
RG EnsemblPlants;
RL Submitted (MAR-2015) to UniProtKB.
CC -!- FUNCTION: This enzyme is required for electron transfer from NADP to
CC cytochrome P450 in microsomes. It can also provide electron transfer to
CC heme oxygenase and cytochrome B5. Reduces a variety of substrates in
CC vitro, such as cytochrome c, feericyanide and dichloroindophenol.
CC {ECO:0000256|ARBA:ARBA00053665}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=2 oxidized [cytochrome P450] + NADPH = 2 reduced [cytochrome
CC P450] + NADP(+) + H(+); Xref=Rhea:RHEA:24040, Rhea:RHEA-COMP:14627,
CC Rhea:RHEA-COMP:14628, ChEBI:CHEBI:15378, ChEBI:CHEBI:55376,
CC ChEBI:CHEBI:57783, ChEBI:CHEBI:58349, ChEBI:CHEBI:60344; EC=1.6.2.4;
CC Evidence={ECO:0000256|HAMAP-Rule:MF_03212,
CC ECO:0000256|PIRNR:PIRNR000208};
CC -!- COFACTOR:
CC Name=FAD; Xref=ChEBI:CHEBI:57692;
CC Evidence={ECO:0000256|HAMAP-Rule:MF_03212};
CC Note=Binds 1 FAD per monomer. {ECO:0000256|HAMAP-Rule:MF_03212};
CC -!- COFACTOR:
CC Name=FMN; Xref=ChEBI:CHEBI:58210;
CC Evidence={ECO:0000256|HAMAP-Rule:MF_03212};
CC Note=Binds 1 FMN per monomer. {ECO:0000256|HAMAP-Rule:MF_03212};
CC -!- SUBCELLULAR LOCATION: Endoplasmic reticulum membrane
CC {ECO:0000256|ARBA:ARBA00004131, ECO:0000256|HAMAP-Rule:MF_03212};
CC Single-pass membrane protein {ECO:0000256|ARBA:ARBA00004131,
CC ECO:0000256|HAMAP-Rule:MF_03212}; Cytoplasmic side
CC {ECO:0000256|ARBA:ARBA00004131, ECO:0000256|HAMAP-Rule:MF_03212}.
CC -!- SIMILARITY: Belongs to the NADPH--cytochrome P450 reductase family.
CC {ECO:0000256|HAMAP-Rule:MF_03212}.
CC -!- SIMILARITY: In the C-terminal section; belongs to the flavoprotein
CC pyridine nucleotide cytochrome reductase family. {ECO:0000256|HAMAP-
CC Rule:MF_03212, ECO:0000256|PIRNR:PIRNR000208}.
CC -!- SIMILARITY: In the N-terminal section; belongs to the flavodoxin
CC family. {ECO:0000256|HAMAP-Rule:MF_03212}.
CC -!- CAUTION: Lacks conserved residue(s) required for the propagation of
CC feature annotation. {ECO:0000256|HAMAP-Rule:MF_03212}.
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DR RefSeq; XP_013598520.1; XM_013743066.1.
DR AlphaFoldDB; A0A0D3DGY4; -.
DR STRING; 109376.A0A0D3DGY4; -.
DR EnsemblPlants; Bo7g114320.1; Bo7g114320.1; Bo7g114320.
DR GeneID; 106306443; -.
DR Gramene; Bo7g114320.1; Bo7g114320.1; Bo7g114320.
DR KEGG; boe:106306443; -.
DR eggNOG; KOG1158; Eukaryota.
DR HOGENOM; CLU_001570_17_3_1; -.
DR OMA; VFTIHAD; -.
DR OrthoDB; 1856718at2759; -.
DR Proteomes; UP000032141; Chromosome C7.
DR GO; GO:0005829; C:cytosol; IEA:TreeGrafter.
DR GO; GO:0005789; C:endoplasmic reticulum membrane; IEA:UniProtKB-SubCell.
DR GO; GO:0050660; F:flavin adenine dinucleotide binding; IEA:UniProtKB-UniRule.
DR GO; GO:0010181; F:FMN binding; IEA:UniProtKB-UniRule.
DR GO; GO:0050661; F:NADP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0003958; F:NADPH-hemoprotein reductase activity; IEA:UniProtKB-UniRule.
DR GO; GO:0009698; P:phenylpropanoid metabolic process; IEA:UniProtKB-KW.
DR CDD; cd06204; CYPOR; 1.
DR FunFam; 1.20.990.10:FF:000003; NADPH--cytochrome P450 reductase; 1.
DR FunFam; 3.40.50.360:FF:000023; NADPH--cytochrome P450 reductase; 1.
DR FunFam; 3.40.50.80:FF:000001; NADPH--cytochrome P450 reductase 1; 1.
DR Gene3D; 3.40.50.360; -; 1.
DR Gene3D; 1.20.990.10; NADPH-cytochrome p450 Reductase, Chain A, domain 3; 1.
DR Gene3D; 3.40.50.80; Nucleotide-binding domain of ferredoxin-NADP reductase (FNR) module; 1.
DR Gene3D; 2.40.30.10; Translation factors; 1.
DR HAMAP; MF_03212; NCPR; 1.
DR InterPro; IPR003097; CysJ-like_FAD-binding.
DR InterPro; IPR017927; FAD-bd_FR_type.
DR InterPro; IPR001094; Flavdoxin-like.
DR InterPro; IPR008254; Flavodoxin/NO_synth.
DR InterPro; IPR001709; Flavoprot_Pyr_Nucl_cyt_Rdtase.
DR InterPro; IPR029039; Flavoprotein-like_sf.
DR InterPro; IPR039261; FNR_nucleotide-bd.
DR InterPro; IPR023173; NADPH_Cyt_P450_Rdtase_alpha.
DR InterPro; IPR001433; OxRdtase_FAD/NAD-bd.
DR InterPro; IPR023208; P450R.
DR InterPro; IPR017938; Riboflavin_synthase-like_b-brl.
DR PANTHER; PTHR19384:SF17; NADPH--CYTOCHROME P450 REDUCTASE; 1.
DR PANTHER; PTHR19384; NITRIC OXIDE SYNTHASE-RELATED; 1.
DR Pfam; PF00667; FAD_binding_1; 1.
DR Pfam; PF00258; Flavodoxin_1; 1.
DR Pfam; PF00175; NAD_binding_1; 1.
DR PIRSF; PIRSF000208; P450R; 1.
DR PRINTS; PR00369; FLAVODOXIN.
DR PRINTS; PR00371; FPNCR.
DR SUPFAM; SSF52343; Ferredoxin reductase-like, C-terminal NADP-linked domain; 1.
DR SUPFAM; SSF52218; Flavoproteins; 1.
DR SUPFAM; SSF63380; Riboflavin synthase domain-like; 1.
DR PROSITE; PS51384; FAD_FR; 1.
DR PROSITE; PS50902; FLAVODOXIN_LIKE; 1.
PE 3: Inferred from homology;
KW Endoplasmic reticulum {ECO:0000256|ARBA:ARBA00022824, ECO:0000256|HAMAP-
KW Rule:MF_03212};
KW FAD {ECO:0000256|ARBA:ARBA00022827, ECO:0000256|HAMAP-Rule:MF_03212};
KW Flavoprotein {ECO:0000256|ARBA:ARBA00022630, ECO:0000256|HAMAP-
KW Rule:MF_03212};
KW FMN {ECO:0000256|ARBA:ARBA00022643, ECO:0000256|HAMAP-Rule:MF_03212};
KW Membrane {ECO:0000256|ARBA:ARBA00023136, ECO:0000256|HAMAP-Rule:MF_03212};
KW NADP {ECO:0000256|ARBA:ARBA00022857, ECO:0000256|HAMAP-Rule:MF_03212};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002, ECO:0000256|HAMAP-
KW Rule:MF_03212}; Phenylpropanoid metabolism {ECO:0000256|ARBA:ARBA00023051};
KW Reference proteome {ECO:0000313|Proteomes:UP000032141};
KW Transmembrane {ECO:0000256|ARBA:ARBA00022692, ECO:0000256|HAMAP-
KW Rule:MF_03212};
KW Transmembrane helix {ECO:0000256|ARBA:ARBA00022989, ECO:0000256|HAMAP-
KW Rule:MF_03212}.
FT TRANSMEM 49..69
FT /note="Helical"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT DOMAIN 104..254
FT /note="Flavodoxin-like"
FT /evidence="ECO:0000259|PROSITE:PS50902"
FT DOMAIN 307..553
FT /note="FAD-binding FR-type"
FT /evidence="ECO:0000259|PROSITE:PS51384"
FT BINDING 110..115
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 165..168
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 203..212
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 238
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 327
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 486..489
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 504..506
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 520..523
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 567
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 628..629
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 634..638
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 670
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
FT BINDING 708
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_03212"
SQ SEQUENCE 708 AA; 78613 MW; 8B2069C4CC6E5CB5 CRC64;

Query Match 99.8%; Score 3670; Length 708;
Best Local Similarity 99.7%;
Matches 706; Conservative 1; Mismatches 1; Indels 0; Gaps 0;

Qy 1 MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAELSSMLIENRQFAMIISTSIAVL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAELSSMLIENRQFAMIISTSIAVL 60

Qy 61 IGCIVMLLWRRSGGSGSSKRAETLKPLVLKPPREDEVDDGRKKVTIFFGTQTGTAEGFAK 120
|||||||||||||||||||| |||||||:|||||||||||||||||||||||||||||||
Db 61 IGCIVMLLWRRSGGSGSSKRVETLKPLVIKPPREDEVDDGRKKVTIFFGTQTGTAEGFAK 120

Qy 121 ALGEEARARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFY 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 ALGEEARARYEKTRFKIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFY 180

Qy 181 KWFTEGDDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVHVGLGDDDQ 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 KWFTEGDDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQGAQRLVHVGLGDDDQ 240

Qy 241 CIEDDFTAWREALWPELDTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKNLANGNG 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 CIEDDFTAWREALWPELDTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKNLANGNG 300

Qy 301 HAVFDAQHPYRANVAVRRELHTPESDRSCTHLEFDIAGSGLTYETGDHVGVLSDNLNETV 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 HAVFDAQHPYRANVAVRRELHTPESDRSCTHLEFDIAGSGLTYETGDHVGVLSDNLNETV 360

Qy 361 EEALRLLDMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTRYACLLSSPKKSALL 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 EEALRLLDMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTRYACLLSSPKKSALL 420

Qy 421 ALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAVA 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 ALAAHASDPTEAERLKHLASPAGKDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAVA 480

Qy 481 PRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKSAVPYEKSENCCS 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 PRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIHKGVCSTWMKSAVPYEKSENCCS 540

Qy 541 APIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFFGCRN 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 APIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVLFFGCRN 600

Qy 601 RRMDFIYEEELQRFLESGALSELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYVYV 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 RRMDFIYEEELQRFLESGALSELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYVYV 660

Qy 661 CGDAKGMARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGRYLRDVW 708
||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 CGDAKGMARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGRYLRDVW 708

Sequence alignment of DFR of SEQ ID NO:58 (“Qy”) and DFR of A0A2U8UVV3 (“Db”)

A0A2U8UVV3_ACEPM
ID A0A2U8UVV3_ACEPM Unreviewed; 360 AA.
AC A0A2U8UVV3;
DT 12-SEP-2018, integrated into UniProtKB/TrEMBL.
DT 12-SEP-2018, sequence version 1.
DT 28-JAN-2026, entry version 16.
DE RecName: Full=Flavanone 4-reductase {ECO:0000256|ARBA:ARBA00042087};
DE EC=1.1.1.219 {ECO:0000256|ARBA:ARBA00039057};
DE EC=1.1.1.234 {ECO:0000256|ARBA:ARBA00039055};
OS Acer palmatum (Japanese maple).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; malvids; Sapindales; Sapindaceae; Hippocastanoideae; Acereae; Acer.
OX NCBI_TaxID=66201 {ECO:0000313|EMBL:AWN08247.1};
RN [1] {ECO:0000313|EMBL:AWN08247.1}
RP NUCLEOTIDE SEQUENCE.
RA Zhong H.Q., Huang M.L., Lin R.Y., Fan R.H.;
RT "Gene cloning of dihydroflavonol 4-reductase (DFR) from Acer palmatum.";
RL Submitted (JUL-2017) to the EMBL/GenBank/DDBJ databases.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(2S)-flavan-4-ol + NADP(+) = (2S)-flavanone + NADPH + H(+);
CC Xref=Rhea:RHEA:11228, ChEBI:CHEBI:15378, ChEBI:CHEBI:15605,
CC ChEBI:CHEBI:15606, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349;
CC EC=1.1.1.234; Evidence={ECO:0000256|ARBA:ARBA00048870};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a (2R,3S,4S)-leucoanthocyanidin + NADP(+) = a (2R,3R)-
CC dihydroflavonol + NADPH + H(+); Xref=Rhea:RHEA:54444,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:138176, ChEBI:CHEBI:138188; EC=1.1.1.219;
CC Evidence={ECO:0000256|ARBA:ARBA00049132};
CC -!- SIMILARITY: Belongs to the NAD(P)-dependent epimerase/dehydratase
CC family. Dihydroflavonol-4-reductase subfamily.
CC {ECO:0000256|ARBA:ARBA00023445}.
CC ---------------------------------------------------------------------------
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DR EMBL; MF460441; AWN08247.1; -; mRNA.
DR AlphaFoldDB; A0A2U8UVV3; -.
DR GO; GO:0045552; F:dihydroflavanol 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0047890; F:flavanone 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0009718; P:anthocyanin-containing compound biosynthetic process; IEA:TreeGrafter.
DR CDD; cd08958; FR_SDR_e; 1.
DR FunFam; 3.40.50.720:FF:000085; Dihydroflavonol reductase; 1.
DR Gene3D; 3.40.50.720; NAD(P)-binding Rossmann-like Domain; 1.
DR InterPro; IPR001509; Epimerase_deHydtase.
DR InterPro; IPR036291; NAD(P)-bd_dom_sf.
DR InterPro; IPR050425; NAD(P)_dehydrat-like.
DR PANTHER; PTHR10366; NAD DEPENDENT EPIMERASE/DEHYDRATASE; 1.
DR PANTHER; PTHR10366:SF564; STEROL-4-ALPHA-CARBOXYLATE 3-DEHYDROGENASE, DECARBOXYLATING; 1.
DR Pfam; PF01370; Epimerase; 1.
DR SUPFAM; SSF51735; NAD(P)-binding Rossmann-fold domains; 1.
PE 2: Evidence at transcript level;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002}.
FT DOMAIN 8..248
FT /note="NAD-dependent epimerase/dehydratase"
FT /evidence="ECO:0000259|Pfam:PF01370"
SQ SEQUENCE 360 AA; 40140 MW; 058E49F2FFD09964 CRC64;

Query Match 100.0%; Score 1891; Length 360;
Best Local Similarity 100.0%;
Matches 360; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVRDPDNEKKVKHLVELPKAKTHLTL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVRDPDNEKKVKHLVELPKAKTHLTL 60

Qy 61 WKADLSDEGSFDEAIHGCTGVFHVATPMDFESKDPENEVIKPTINGVLGIMKACKKAKTV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 WKADLSDEGSFDEAIHGCTGVFHVATPMDFESKDPENEVIKPTINGVLGIMKACKKAKTV 120

Qy 121 KRLVFTSSAGTVDVEEHKKPVYDENSWSDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 KRLVFTSSAGTVDVEEHKKPVYDENSWSDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEE 180

Qy 181 NSIDFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAIIKQGNYVHLDDLCMGHIF 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 NSIDFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAIIKQGNYVHLDDLCMGHIF 240

Qy 241 LYEHAESKGRYFCSSHSATILELSKFLRERYPEYDLPTEYKGVDDSLENVVFCSKKILDL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LYEHAESKGRYFCSSHSATILELSKFLRERYPEYDLPTEYKGVDDSLENVVFCSKKILDL 300

Qy 301 GFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHVAAKGLIPNNSDEIHVAAAEKTTATA 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 GFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHVAAKGLIPNNSDEIHVAAAEKTTATA 360

Sequence alignment of DFR of SEQ ID NO:59 (“Qy”) and DFR of XP_027329642.1 (“Db”)

A0A8B8JE85_ABRPR
ID A0A8B8JE85_ABRPR Unreviewed; 337 AA.
AC A0A8B8JE85;
DT 19-JAN-2022, integrated into UniProtKB/TrEMBL.
DT 19-JAN-2022, sequence version 1.
DT 28-JAN-2026, entry version 18.
DE RecName: Full=Flavanone 4-reductase {ECO:0000256|ARBA:ARBA00042087};
DE EC=1.1.1.219 {ECO:0000256|ARBA:ARBA00039057};
DE EC=1.1.1.234 {ECO:0000256|ARBA:ARBA00039055};
GN Name=LOC113846034 {ECO:0000313|RefSeq:XP_027329642.1};
OS Abrus precatorius (Indian licorice) (Glycine abrus).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; fabids; Fabales; Fabaceae; Papilionoideae; 50 kb inversion clade;
OC NPAAA clade; indigoferoid/millettioid clade; Abreae; Abrus.
OX NCBI_TaxID=3816 {ECO:0000313|Proteomes:UP000694853, ECO:0000313|RefSeq:XP_027329642.1};
RN [1] {ECO:0000313|Proteomes:UP000694853}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RX PubMed=31775284; DOI=10.3390/toxins11120691;
RA Hovde B.T., Daligault H.E., Hanschen E.R., Kunde Y.A., Johnson M.B.,
RA Starkenburg S.R., Johnson S.L.;
RT "Detection of Abrin-Like and Prepropulchellin-Like Toxin Genes and
RT Transcripts Using Whole Genome Sequencing and Full-Length Transcript
RT Sequencing of Abrus precatorius.";
RL Toxins 11:0-0(2019).
RN [2] {ECO:0000313|RefSeq:XP_027329642.1}
RP IDENTIFICATION.
RC TISSUE=Young leaves {ECO:0000313|RefSeq:XP_027329642.1};
RG RefSeq;
RL Submitted (AUG-2025) to UniProtKB.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(2S)-flavan-4-ol + NADP(+) = (2S)-flavanone + NADPH + H(+);
CC Xref=Rhea:RHEA:11228, ChEBI:CHEBI:15378, ChEBI:CHEBI:15605,
CC ChEBI:CHEBI:15606, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349;
CC EC=1.1.1.234; Evidence={ECO:0000256|ARBA:ARBA00048870};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a (2R,3S,4S)-leucoanthocyanidin + NADP(+) = a (2R,3R)-
CC dihydroflavonol + NADPH + H(+); Xref=Rhea:RHEA:54444,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:138176, ChEBI:CHEBI:138188; EC=1.1.1.219;
CC Evidence={ECO:0000256|ARBA:ARBA00049132};
CC -!- SIMILARITY: Belongs to the NAD(P)-dependent epimerase/dehydratase
CC family. Dihydroflavonol-4-reductase subfamily.
CC {ECO:0000256|ARBA:ARBA00023445}.
CC ---------------------------------------------------------------------------
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CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
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DR RefSeq; XP_027329642.1; XM_027473841.1.
DR AlphaFoldDB; A0A8B8JE85; -.
DR GeneID; 113846034; -.
DR KEGG; aprc:113846034; -.
DR OrthoDB; 2735536at2759; -.
DR Proteomes; UP000694853; Unplaced.
DR GO; GO:0045552; F:dihydroflavanol 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0047890; F:flavanone 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0009718; P:anthocyanin-containing compound biosynthetic process; IEA:TreeGrafter.
DR CDD; cd08958; FR_SDR_e; 1.
DR FunFam; 3.40.50.720:FF:000085; Dihydroflavonol reductase; 1.
DR Gene3D; 3.40.50.720; NAD(P)-binding Rossmann-like Domain; 1.
DR InterPro; IPR001509; Epimerase_deHydtase.
DR InterPro; IPR036291; NAD(P)-bd_dom_sf.
DR InterPro; IPR050425; NAD(P)_dehydrat-like.
DR PANTHER; PTHR10366; NAD DEPENDENT EPIMERASE/DEHYDRATASE; 1.
DR PANTHER; PTHR10366:SF564; STEROL-4-ALPHA-CARBOXYLATE 3-DEHYDROGENASE, DECARBOXYLATING; 1.
DR Pfam; PF01370; Epimerase; 1.
DR SUPFAM; SSF51735; NAD(P)-binding Rossmann-fold domains; 1.
PE 3: Inferred from homology;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002};
KW Reference proteome {ECO:0000313|Proteomes:UP000694853}.
FT DOMAIN 8..248
FT /note="NAD-dependent epimerase/dehydratase"
FT /evidence="ECO:0000259|Pfam:PF01370"
SQ SEQUENCE 337 AA; 37763 MW; 95EC355123495CBC CRC64;

Query Match 100.0%; Score 1770; Length 337;
Best Local Similarity 100.0%;
Matches 337; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKVKHLLELPNAKTNLSL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKVKHLLELPNAKTNLSL 60

Qy 61 WKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDIMKACMKAKTV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 WKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDIMKACMKAKTV 120

Qy 121 RRLVFTSSAGTVDVTEHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 RRLVFTSSAGTVDVTEHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE 180

Qy 181 NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQGQFVHLDDLCLAHIF 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQGQFVHLDDLCLAHIF 240

Qy 241 LFQHPKAQGRYICCSHEATIHDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LFQHPKAQGRYICCSHEATIHDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL 300

Qy 301 GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK 337
|||||||||||||||||||||||||||||||||||||
Db 301 GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK 337

Sequence alignment of DFR of SEQ ID NO:60 (“Qy”) and DFR of F5A636 (“Db”)

F5A636_DENMN
ID F5A636_DENMN Unreviewed; 351 AA.
AC F5A636;
DT 28-JUN-2011, integrated into UniProtKB/TrEMBL.
DT 28-JUN-2011, sequence version 1.
DT 28-JAN-2026, entry version 31.
DE RecName: Full=Flavanone 4-reductase {ECO:0000256|ARBA:ARBA00042087};
DE EC=1.1.1.219 {ECO:0000256|ARBA:ARBA00039057};
DE EC=1.1.1.234 {ECO:0000256|ARBA:ARBA00039055};
GN Name=DFR {ECO:0000313|EMBL:AEB96144.1};
OS Dendrobium moniliforme (Orchid) (Epidendrum moniliforme).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; Liliopsida; Asparagales; Orchidaceae;
OC Epidendroideae; Malaxideae; Dendrobiinae; Dendrobium.
OX NCBI_TaxID=142614 {ECO:0000313|EMBL:AEB96144.1};
RN [1] {ECO:0000313|EMBL:AEB96144.1}
RP NUCLEOTIDE SEQUENCE.
RA Koo J.C.;
RT "Molecular analysis of anthocyanin biosynthetic genes from Dendrobium
RT moniliforme.";
RL Submitted (OCT-2010) to the EMBL/GenBank/DDBJ databases.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(2S)-flavan-4-ol + NADP(+) = (2S)-flavanone + NADPH + H(+);
CC Xref=Rhea:RHEA:11228, ChEBI:CHEBI:15378, ChEBI:CHEBI:15605,
CC ChEBI:CHEBI:15606, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349;
CC EC=1.1.1.234; Evidence={ECO:0000256|ARBA:ARBA00048870};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a (2R,3S,4S)-leucoanthocyanidin + NADP(+) = a (2R,3R)-
CC dihydroflavonol + NADPH + H(+); Xref=Rhea:RHEA:54444,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:138176, ChEBI:CHEBI:138188; EC=1.1.1.219;
CC Evidence={ECO:0000256|ARBA:ARBA00049132};
CC -!- SIMILARITY: Belongs to the NAD(P)-dependent epimerase/dehydratase
CC family. Dihydroflavonol-4-reductase subfamily.
CC {ECO:0000256|ARBA:ARBA00023445}.
CC ---------------------------------------------------------------------------
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CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR EMBL; HQ412559; AEB96144.1; -; mRNA.
DR AlphaFoldDB; F5A636; -.
DR GO; GO:0045552; F:dihydroflavanol 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0047890; F:flavanone 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0009813; P:flavonoid biosynthetic process; IEA:UniProtKB-KW.
DR CDD; cd08958; FR_SDR_e; 1.
DR FunFam; 3.40.50.720:FF:000085; Dihydroflavonol reductase; 1.
DR Gene3D; 3.40.50.720; NAD(P)-binding Rossmann-like Domain; 1.
DR InterPro; IPR001509; Epimerase_deHydtase.
DR InterPro; IPR036291; NAD(P)-bd_dom_sf.
DR InterPro; IPR050425; NAD(P)_dehydrat-like.
DR PANTHER; PTHR10366; NAD DEPENDENT EPIMERASE/DEHYDRATASE; 1.
DR PANTHER; PTHR10366:SF564; STEROL-4-ALPHA-CARBOXYLATE 3-DEHYDROGENASE, DECARBOXYLATING; 1.
DR Pfam; PF01370; Epimerase; 1.
DR SUPFAM; SSF51735; NAD(P)-binding Rossmann-fold domains; 1.
PE 2: Evidence at transcript level;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002}.
FT DOMAIN 9..250
FT /note="NAD-dependent epimerase/dehydratase"
FT /evidence="ECO:0000259|Pfam:PF01370"
SQ SEQUENCE 351 AA; 39588 MW; D2AAF057C1893507 CRC64;

Query Match 100.0%; Score 1827; Length 351;
Best Local Similarity 100.0%;
Matches 351; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRATVRDPTNLKKVKPLLDLPRSNELLS 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRATVRDPTNLKKVKPLLDLPRSNELLS 60

Qy 61 IWKADLDGIEGSFDEVIRGSIGVFHVATPMNFQSKDPENEVIQPAINGLLGILRSCKNAG 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 IWKADLDGIEGSFDEVIRGSIGVFHVATPMNFQSKDPENEVIQPAINGLLGILRSCKNAG 120

Qy 121 SVQRVIFTSSAGTVNVEEHQAAAYDETCWSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFV 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 SVQRVIFTSSAGTVNVEEHQAAAYDETCWSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFV 180

Qy 181 KDNHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHYSILKQIQFVHLDDLCDAH 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 KDNHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHYSILKQIQFVHLDDLCDAH 240

Qy 241 IFLFEHPKANGRYICSSYDSTIYGLAEMLKNRYPTYAIPHKFKEIDPDIKCVSFSSKKLM 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 IFLFEHPKANGRYICSSYDSTIYGLAEMLKNRYPTYAIPHKFKEIDPDIKCVSFSSKKLM 300

Qy 301 ELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVLAAEKFEEVKEQIAVK 351
|||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 ELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVLAAEKFEEVKEQIAVK 351

Sequence alignment of DFR of SEQ ID NO:61 (“Qy”) and DFR of A0A2P6PYS2 (“Db”)

A0A2P6PYS2_ROSCH
ID A0A2P6PYS2_ROSCH Unreviewed; 349 AA.
AC A0A2P6PYS2;
DT 23-MAY-2018, integrated into UniProtKB/TrEMBL.
DT 23-MAY-2018, sequence version 1.
DT 28-JAN-2026, entry version 26.
DE RecName: Full=Flavanone 4-reductase {ECO:0000256|ARBA:ARBA00042087};
DE EC=1.1.1.219 {ECO:0000256|ARBA:ARBA00039057};
DE EC=1.1.1.234 {ECO:0000256|ARBA:ARBA00039055};
GN ORFNames=RchiOBHm_Chr6g0301421 {ECO:0000313|EMBL:PRQ27072.1};
OS Rosa chinensis (China rose).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC rosids; fabids; Rosales; Rosaceae; Rosoideae; Rosoideae incertae sedis;
OC Rosa.
OX NCBI_TaxID=74649 {ECO:0000313|EMBL:PRQ27072.1, ECO:0000313|Proteomes:UP000238479};
RN [1] {ECO:0000313|EMBL:PRQ27072.1, ECO:0000313|Proteomes:UP000238479}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=cv. Old Blush {ECO:0000313|Proteomes:UP000238479};
RA Bendahmane M.;
RT "The Rosa genome provides new insights in the design of modern roses.";
RL Nat. Genet. 0:0-0(2018).
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(2S)-flavan-4-ol + NADP(+) = (2S)-flavanone + NADPH + H(+);
CC Xref=Rhea:RHEA:11228, ChEBI:CHEBI:15378, ChEBI:CHEBI:15605,
CC ChEBI:CHEBI:15606, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349;
CC EC=1.1.1.234; Evidence={ECO:0000256|ARBA:ARBA00048870};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a (2R,3S,4S)-leucoanthocyanidin + NADP(+) = a (2R,3R)-
CC dihydroflavonol + NADPH + H(+); Xref=Rhea:RHEA:54444,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:138176, ChEBI:CHEBI:138188; EC=1.1.1.219;
CC Evidence={ECO:0000256|ARBA:ARBA00049132};
CC -!- SIMILARITY: Belongs to the NAD(P)-dependent epimerase/dehydratase
CC family. Dihydroflavonol-4-reductase subfamily.
CC {ECO:0000256|ARBA:ARBA00023445}.
CC -!- CAUTION: The sequence shown here is derived from an EMBL/GenBank/DDBJ
CC whole genome shotgun (WGS) entry which is preliminary data.
CC {ECO:0000313|EMBL:PRQ27072.1}.
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DR EMBL; PDCK01000044; PRQ27072.1; -; Genomic_DNA.
DR AlphaFoldDB; A0A2P6PYS2; -.
DR SMR; A0A2P6PYS2; -.
DR STRING; 74649.A0A2P6PYS2; -.
DR Gramene; PRQ27072; PRQ27072; RchiOBHm_Chr6g0301421.
DR OMA; ETCWSDV; -.
DR OrthoDB; 2735536at2759; -.
DR Proteomes; UP000238479; Chromosome 6.
DR GO; GO:0045552; F:dihydroflavanol 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0047890; F:flavanone 4-reductase activity; IEA:UniProtKB-EC.
DR GO; GO:0009718; P:anthocyanin-containing compound biosynthetic process; IEA:TreeGrafter.
DR CDD; cd08958; FR_SDR_e; 1.
DR FunFam; 3.40.50.720:FF:000085; Dihydroflavonol reductase; 1.
DR Gene3D; 3.40.50.720; NAD(P)-binding Rossmann-like Domain; 1.
DR InterPro; IPR001509; Epimerase_deHydtase.
DR InterPro; IPR036291; NAD(P)-bd_dom_sf.
DR InterPro; IPR050425; NAD(P)_dehydrat-like.
DR PANTHER; PTHR10366; NAD DEPENDENT EPIMERASE/DEHYDRATASE; 1.
DR PANTHER; PTHR10366:SF564; STEROL-4-ALPHA-CARBOXYLATE 3-DEHYDROGENASE, DECARBOXYLATING; 1.
DR Pfam; PF01370; Epimerase; 1.
DR SUPFAM; SSF51735; NAD(P)-binding Rossmann-fold domains; 1.
PE 3: Inferred from homology;
KW Flavonoid biosynthesis {ECO:0000256|ARBA:ARBA00023241};
KW NADP {ECO:0000256|ARBA:ARBA00022857};
KW Oxidoreductase {ECO:0000256|ARBA:ARBA00023002,
KW ECO:0000313|EMBL:PRQ27072.1};
KW Reference proteome {ECO:0000313|Proteomes:UP000238479}.
FT DOMAIN 8..248
FT /note="NAD-dependent epimerase/dehydratase"
FT /evidence="ECO:0000259|Pfam:PF01370"
SQ SEQUENCE 349 AA; 38942 MW; 085EC711F7E0271B CRC64;

Query Match 100.0%; Score 1830; Length 349;
Best Local Similarity 100.0%;
Matches 349; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKVKHLLDLPKAATHLTL 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKVKHLLDLPKAATHLTL 60

Qy 61 WKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDIMKACLKAKTV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 WKADLAEEGSFDEAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDIMKACLKAKTV 120

Qy 121 RRLVFTASAGSVNVEETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 RRLVFTASAGSVNVEETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE 180

Qy 181 NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQGQFIHLDDLCQSHIY 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQGQFIHLDDLCQSHIY 240

Qy 241 LYEHPKAEGRYICSSHDATIHEIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLET 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 LYEHPKAEGRYICSSHDATIHEIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLET 300

Qy 301 GFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG 349
|||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 GFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG 349

Sequence alignment of the CPR of SEQ ID NO:53 (“Qy”) and CPR of SEQ ID NO:54 (“Db”)

Title: US-17-720-020-53
Perfect score: 5219
Sequence: 1 MASNSNLIRAIESALGVSFG..........KAEVIVKKLQMEGRYLRDVW 1007

Scoring table: BLOSUM62
Gapop 10.0 , Gapext 0.5

Searched: 1 seqs, 1007 residues

Total number of hits satisfying chosen parameters: 1

Minimum DB seq length: 0
Maximum DB seq length: inf

Post-processing: Minimum Match 0%
Maximum Match 100%
Listing first 50 summaries

Database : US-17-720-020-54.fasta:*

SUMMARIES
%
Result Query
No. Score Match Length DB ID Description
----------------------------------------------------------------------------
1 5219 100.0 1007 1 US-17-720-020-54 FLAVONOID AND ANTH

ALIGNMENTS

RESULT 1
US-17-720-020-54

Query Match 100.0%; Score 5219; DB 1; Length 1007;
Best Local Similarity 100.0%;
Matches 1007; Conservative 0; Mismatches 0; Indels 0; Gaps 0;

Qy 1 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPV 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPV 60

Qy 61 VISKPLLVEEEEEEDEVEAGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEKAIVKVV 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 VISKPLLVEEEEEEDEVEAGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEKAIVKVV 120

Qy 121 DLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 DLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY 180

Qy 181 GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELD 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDDFAAWRETLWPELD 240

Qy 241 QLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSN 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 QLLRDEDDANTVSTPYAAAIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREGSN 300

Qy 301 QQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 QQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF 360

Qy 361 LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEE 420
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 361 LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFAKSLAEE 420

Qy 421 IKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTE 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 IKARYEKAIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYGDGEPTDNAARFYKWFTE 480

Qy 481 ENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 ENERGAWLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD 540

Qy 541 FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNA 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMANGNA 600

Qy 601 SYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEE 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 SYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDISGTGIIYETGDHVGVYADNFDEVVEE 660

Qy 661 AANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 AANLLGQPLELLFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA 720

Qy 721 LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAP 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAP 780

Qy 781 RLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSA 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 RLQPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGVCSTWMKNAVPLEKSHDCSSA 840

Qy 841 PIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 PIFTRTSNFKLPTDPSIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR 900

Qy 901 RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVC 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAAQVWGLISQGGYLYVC 960

Qy 961 GDAKGMARDVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLRDVW 1007
|||||||||||||||||||||||||||||||||||||||||||||||
Db 961 GDAKGMARDVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLRDVW 1007

Prosecution Timeline

• Apr 13, 2022: Application Filed
• Sep 01, 2023: Non-Final Rejection — §103
• Mar 07, 2024: Response Filed
• May 18, 2024: Final Rejection — §103
• Nov 22, 2024: Request for Continued Examination
• Nov 27, 2024: Response after Non-Final Action
• Mar 09, 2025: Non-Final Rejection — §103
• Sep 09, 2025: Response Filed
• Oct 28, 2025: Final Rejection — §103
• Feb 13, 2026: Request for Continued Examination
• Feb 18, 2026: Response after Non-Final Action
• Apr 06, 2026: Non-Final Rejection — §103 (current)