DETAILED ACTION
Status of the Application
Claims 1-2, 7, 10, 12, 14-16, 24, 27, 34, 44, 49, 51, 53, 56, 59, 63, 66 are pending.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment of claims 1-2, 7 and 10 as submitted in a communication filed on 3/16/2026 is acknowledged.
Applicant elected with traverse Group II, claims 1-2, 7, 10, drawn in part to a variant of the polypeptide of SEQ ID NO: 153, in a communication filed on 10/7/2025, and elected without traverse the amino acid position N335 and the substitution N335D, in a communication filed on 11/21/2025.
Claims 12, 14-16, 24, 27, 34, 44, 49, 51, 53, 56, 59, 63, 66 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 10/7/2025.
Claims 1-2, 7 and 10 are at issue and will be examined only to the extent they encompass the elected invention.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claim Objections
Claim 1 is objected to due to the recitation of “wherein the one or more amino acid alterations are at one or more positions corresponding to positions in SEQ ID NO: 153....and selected from positions…335…, and wherein the engineered CAR shares at least 85% sequence identity to SEQ ID NO: 153..optionally wherein the one or more amino acid alterations is an amino acid substitution”. To enhance clarity, the term should be amended to recite “wherein the one or more amino acid alterations are at one or more positions corresponding to positions in SEQ ID NO: 153.. selected from positions…335…, and wherein the engineered CAR has at least 85% sequence identity to the polypeptide of SEQ ID NO: 153..optionally wherein the one or more amino acid alterations are amino acid substitutions”. Appropriate correction is required.
Claim 10 is objected to due to the recitation of “E” twice in part (a). Appropriate correction is required.
Claims 1-2, 7 and 10 are objected for being directed in part to non-elected inventions (Groups I, III). Appropriate correction is required.
Claim Rejections - 35 USC § 101
Claims 1, 2, 7 and 10 were rejected under 35 U.S.C. 101 because the claimed invention was directed to products of nature without significantly more.
Applicant argues that the claims as currently presented recite that the engineered CAR shares at least 85% sequence identity to SEQ ID NO: 153, 152 or 254 and that the cited references require a lower sequence identity.
Applicant’s arguments have been fully considered. In view of Applicant’s amendment of claim 1, which now requires the carboxylic acid reductase to comprise at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, and the fact that the CAR enzymes of Tarcisio et al., Amande et al. and Jude et al do not meet the recited % sequence identity, this rejection is hereby withdrawn. A new rejection follows.
Claims 1, 2, 7 and 10 are rejected under 35 U.S.C. 101 because the claimed invention was directed to products of nature without significantly more. New grounds of rejection are necessitated by amendment.
The claims recite in part a protein which is a variant of the polypeptide of SEQ ID NO: 153 having at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, wherein said protein comprises one or more substitution at positions corresponding to positions 299, 391, 275 and/or 812 of the polypeptide of SEQ ID NO: 153, wherein said variant can comprise an E, D, P, V, C, L, G, A, M, Q, T, Y, N, W, S, I or F at the positions corresponding to positions 299, 391, 275 and/or 812 of the polypeptide of SEQ ID NO: 153. It should be noted that the protein of claims 1, 2, 7 and 10 is not limited with regard to additional mutations with respect to the polypeptide of SEQ ID NO: 153. All that is required is that the protein claimed has at least one of the recited substitution. See Claim Rejections under 35 USC § 112(b) or Second Paragraph (pre-AIA ) below for claim interpretation. Thus, a protein that comprises the recited substitution as well as additional modifications is also encompassed by the claims.
This judicial exception is not integrated into a practical application because the claims are directed to a naturally occurring protein as evidenced by Li et al. (GenBank accession No. AAS03357 1/30/2004), Tortoli et al. (GenBank accession No. ORB75922 4/11/2017) and Gonzalez-Perez et al. (GenBank accession No. EJO87171 8/13/2012). The claims do not include additional elements that are sufficient to amount to significantly more than the judicial exception because claims 1, 2, 7 and 10 fully encompass a naturally-occurring protein. As shown in the alignment below, Li et al. teach a Mycobacterium paratuberculosis carboxylic acid reductase (CAR) that comprises a substitution at a position corresponding to position 391 of the polypeptide of SEQ ID NO: 153, wherein said substitution is with a glycine. The CAR of Li et al. comprises 99.9% sequence identity to the polypeptide of SEQ ID NO: 153 (99.9% = 1172x100/1173; SEQ ID NO: 153 has 1173 amino acids). As shown in the alignment below, Tortoli et al. teach a Mycobacterium scrofulaceum carboxylic acid reductase (CAR) that comprises substitutions at positions corresponding to positions 299 and 391 of the polypeptide of SEQ ID NO: 153, wherein said substitutions are with a glycine, and further comprises substitutions at positions 275 and 812 of the polypeptide of SEQ ID NO: 153. The CAR of Tortoli et al. comprises 85% sequence identity to the polypeptide of SEQ ID NO: 153 (85% = 996x100/1173; SEQ ID NO: 153 has 1173 amino acids). As shown in the alignment below, Gonzalez-Perez et al. teach a Mycobacterium colombiense carboxylic acid reductase (CAR) that comprises substitutions at positions corresponding to positions 299 and 391 of the polypeptide of SEQ ID NO: 153, wherein said substitutions are with a glycine. The CAR of Gonzalez-Perez et al. comprises 90% sequence identity to the polypeptide of SEQ ID NO: 153 (90% = 1051x100/1173; SEQ ID NO: 153 has 1173 amino acids). See underlined/bold/italicized residues. Please note that in the absence of any additional distinguishing feature not naturally found in a protein, the term “engineered” is a product-by-process limitation which does not convey any structural and/or functional differences between a man-made and a naturally-occurring product. With regard to the specific functional characteristics recited in claim 2, it is noted that the specification asserts that a variant of the polypeptide of SEQ ID NO: 153 having the recited substitution would have those functional characteristics. Therefore, it follows that the CAR enzymes of Li et al, Tortoli et al. and Gonzalez-Perez et al. also comprise those functional characteristics. As such, the claims encompass subject matter which is not patent-eligible.
SEQ ID NO: 153
RESULT 1
Q741P9_MYCPA
ID Q741P9_MYCPA Unreviewed; 1173 AA.
AC Q741P9;
DT 05-JUL-2004, integrated into UniProtKB/TrEMBL.
DT 05-JUL-2004, sequence version 1.
DT 08-OCT-2025, entry version 117.
DE RecName: Full=Carboxylic acid reductase {ECO:0000256|HAMAP-Rule:MF_02247};
DE Short=CAR {ECO:0000256|HAMAP-Rule:MF_02247};
DE EC=1.2.1.- {ECO:0000256|HAMAP-Rule:MF_02247};
DE AltName: Full=ATP/NADPH-dependent carboxylic acid reductase {ECO:0000256|HAMAP-Rule:MF_02247};
GN Name=fadD9 {ECO:0000313|EMBL:AAS03357.1};
GN Synonyms=car {ECO:0000256|HAMAP-Rule:MF_02247};
GN OrderedLocusNames=MAP_1040c {ECO:0000313|EMBL:AAS03357.1};
OS Mycolicibacterium paratuberculosis (strain ATCC BAA-968 / K-10)
OS (Mycobacterium paratuberculosis).
OC Bacteria; Bacillati; Actinomycetota; Actinomycetes; Mycobacteriales;
OC Mycobacteriaceae; Mycobacterium; Mycobacterium avium complex (MAC).
OX NCBI_TaxID=262316 {ECO:0000313|EMBL:AAS03357.1, ECO:0000313|Proteomes:UP000000580};
RN [1] {ECO:0000313|EMBL:AAS03357.1, ECO:0000313|Proteomes:UP000000580}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC BAA-968 / K-10 {ECO:0000313|Proteomes:UP000000580};
RX PubMed=16116077; DOI=10.1073/pnas.0505662102;
RA Li L., Bannantine J.P., Zhang Q., Amonsin A., May B.J., Alt D., Banerji N.,
RA Kanjilal S., Kapur V.;
RT "The complete genome sequence of Mycobacterium avium subspecies
RT paratuberculosis.";
RL Proc. Natl. Acad. Sci. U.S.A. 102:12344-12349(2005).
CC -!- FUNCTION: Catalyzes the ATP- and NADPH-dependent reduction of
CC carboxylic acids to the corresponding aldehydes. {ECO:0000256|HAMAP-
CC Rule:MF_02247}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a carboxylate + ATP + NADPH + H(+) = an aldehyde + AMP +
CC diphosphate + NADP(+); Xref=Rhea:RHEA:50916, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:17478, ChEBI:CHEBI:29067, ChEBI:CHEBI:30616,
CC ChEBI:CHEBI:33019, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:456215; Evidence={ECO:0000256|HAMAP-Rule:MF_02247};
CC -!- COFACTOR:
CC Name=pantetheine 4'-phosphate; Xref=ChEBI:CHEBI:47942;
CC Evidence={ECO:0000256|HAMAP-Rule:MF_02247};
CC Note=Binds 1 phosphopantetheine covalently. {ECO:0000256|HAMAP-
CC Rule:MF_02247};
CC -!- DOMAIN: The N-terminal domain likely catalyzes substrate activation by
CC formation of an initial acyl-AMP intermediate, the central region
CC contains the phosphopantetheine attachment site, and the C-terminal
CC domain catalyzes the reduction by NADPH of the intermediate thioester
CC formed from the attack of the phosphopantetheine thiol at the carbonyl
CC carbon of acyl-AMP. {ECO:0000256|HAMAP-Rule:MF_02247}.
CC -!- SIMILARITY: Belongs to the ATP-dependent AMP-binding enzyme family.
CC Carboxylic acid reductase subfamily. {ECO:0000256|HAMAP-Rule:MF_02247}.
CC -!- CAUTION: Lacks conserved residue(s) required for the propagation of
CC feature annotation. {ECO:0000256|HAMAP-Rule:MF_02247}.
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DR EMBL; AE016958; AAS03357.1; -; Genomic_DNA.
DR RefSeq; WP_003872682.1; NZ_CP106873.1.
DR AlphaFoldDB; Q741P9; -.
DR SMR; Q741P9; -.
DR STRING; 262316.MAP_1040c; -.
DR KEGG; mpa:MAP_1040c; -.
DR PATRIC; fig|262316.17.peg.1093; -.
DR eggNOG; COG1022; Bacteria.
DR eggNOG; COG3320; Bacteria.
DR HOGENOM; CLU_009549_0_0_11; -.
DR Proteomes; UP000000580; Chromosome.
DR GO; GO:0016020; C:membrane; IEA:TreeGrafter.
DR GO; GO:0005524; F:ATP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0004467; F:long-chain fatty acid-CoA ligase activity; IEA:TreeGrafter.
DR GO; GO:0050661; F:NADP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0016620; F:oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor; IEA:UniProtKB-UniRule.
DR GO; GO:0031177; F:phosphopantetheine binding; IEA:UniProtKB-UniRule.
DR CDD; cd17632; AFD_CAR-like; 1.
DR CDD; cd05235; SDR_e1; 1.
DR Gene3D; 1.10.1200.10; ACP-like; 1.
DR Gene3D; 3.40.50.12780; N-terminal domain of ligase-like; 1.
DR Gene3D; 3.40.50.720; NAD(P)-binding Rossmann-like Domain; 1.
DR HAMAP; MF_02247; Carbox_acid_reduct; 1.
DR InterPro; IPR036736; ACP-like_sf.
DR InterPro; IPR020845; AMP-binding_CS.
DR InterPro; IPR000873; AMP-dep_synth/lig_dom.
DR InterPro; IPR042099; ANL_N_sf.
DR InterPro; IPR046407; CAR.
DR InterPro; IPR013120; Far_NAD-bd.
DR InterPro; IPR036291; NAD(P)-bd_dom_sf.
DR InterPro; IPR020806; PKS_PP-bd.
DR InterPro; IPR009081; PP-bd_ACP.
DR InterPro; IPR010080; Thioester_reductase-like_dom.
DR NCBIfam; NF041592; carboxyl_red; 1.
DR NCBIfam; TIGR01746; Thioester-redct; 1.
DR PANTHER; PTHR43272:SF33; AMP-BINDING DOMAIN-CONTAINING PROTEIN-RELATED; 1.
DR PANTHER; PTHR43272; LONG-CHAIN-FATTY-ACID--COA LIGASE; 1.
DR Pfam; PF00501; AMP-binding; 1.
DR Pfam; PF07993; NAD_binding_4; 1.
DR Pfam; PF00550; PP-binding; 1.
DR SMART; SM00823; PKS_PP; 1.
DR SUPFAM; SSF56801; Acetyl-CoA synthetase-like; 1.
DR SUPFAM; SSF47336; ACP-like; 1.
DR SUPFAM; SSF51735; NAD(P)-binding Rossmann-fold domains; 1.
DR PROSITE; PS00455; AMP_BINDING; 1.
DR PROSITE; PS50075; CARRIER; 1.
PE 3: Inferred from homology;
KW ATP-binding {ECO:0000256|ARBA:ARBA00022840, ECO:0000256|HAMAP-
KW Rule:MF_02247}; NADP {ECO:0000256|HAMAP-Rule:MF_02247};
KW Nucleotide-binding {ECO:0000256|ARBA:ARBA00022741, ECO:0000256|HAMAP-
KW Rule:MF_02247}; Oxidoreductase {ECO:0000256|HAMAP-Rule:MF_02247};
KW Phosphopantetheine {ECO:0000256|ARBA:ARBA00022450, ECO:0000256|HAMAP-
KW Rule:MF_02247};
KW Phosphoprotein {ECO:0000256|ARBA:ARBA00022553, ECO:0000256|HAMAP-
KW Rule:MF_02247}; Reference proteome {ECO:0000313|Proteomes:UP000000580}.
FT DOMAIN 651..726
FT /note="Carrier"
FT /evidence="ECO:0000259|PROSITE:PS50075"
FT BINDING 294
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 392
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 413..414
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 418
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 491
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 503..506
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 512
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 612
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 783..786
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 810
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 820
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 850..851
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 876..878
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 916
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 952
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 956
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT MOD_RES 685
FT /note="O-(pantetheine 4'-phosphoryl)serine"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
SQ SEQUENCE 1173 AA; 127969 MW; E33B6F06C1F758E6 CRC64;
Query Match 99.9%; Score 5969; Length 1173;
Best Local Similarity 99.9%;
Matches 1172; Conservative 0; Mismatches 1; Indels 0; Gaps 0;
Qy 1 MSTATHDERLDRRVHELIATDPQFAAAQPDPAITAALEQPGLRLPQIIRTVLDGYADRPA 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1 MSTATHDERLDRRVHELIATDPQFAAAQPDPAITAALEQPGLRLPQIIRTVLDGYADRPA 60
Qy 61 LGQRVVEFVTDAKTGRTSAQLLPRFETITYSEVAQRVSALGRALSDDAVHPGDRVCVLGF 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 61 LGQRVVEFVTDAKTGRTSAQLLPRFETITYSEVAQRVSALGRALSDDAVHPGDRVCVLGF 120
Qy 121 NSVDYATIDMALGAIGAVSVPLQTSAAISSLQPIVAETEPTLIASSVNQLSDAVQLITGA 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 121 NSVDYATIDMALGAIGAVSVPLQTSAAISSLQPIVAETEPTLIASSVNQLSDAVQLITGA 180
Qy 181 EQAPTRLVVFDYHPQVDDQREAVQDAAARLSSTGVAVQTLAELLERGKDLPAVAEPPADE 240
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 181 EQAPTRLVVFDYHPQVDDQREAVQDAAARLSSTGVAVQTLAELLERGKDLPAVAEPPADE 240
Qy 241 DSLALLIYTSGSTGAPKGAMYPQSNVGKMWRRGSKNWFGESAASITLNFMPMSHVMGRSI 300
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 241 DSLALLIYTSGSTGAPKGAMYPQSNVGKMWRRGSKNWFGESAASITLNFMPMSHVMGRSI 300
Qy 301 LYGTLGNGGTAYFAARSDLSTLLEDLELVRPTELNFVPRIWETLYGEFQRQVERRLSEAG 360
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 301 LYGTLGNGGTAYFAARSDLSTLLEDLELVRPTELNFVPRIWETLYGEFQRQVERRLSEAG 360
Qy 361 DAGERRAVEAEVLAEQRQYLLGGRFTFAMTSSAPISPELRNWVESLLEMHLMDGYGSTEA 420
|||||||||||||||||||||||||||||| |||||||||||||||||||||||||||||
Db 361 DAGERRAVEAEVLAEQRQYLLGGRFTFAMTGSAPISPELRNWVESLLEMHLMDGYGSTEA 420
Qy 421 GMVLFDGEIQRPPVIDYKLVDVPDLGYFSTDRPHPRGELLLRTENMFPGYYKRAETTAGV 480
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 421 GMVLFDGEIQRPPVIDYKLVDVPDLGYFSTDRPHPRGELLLRTENMFPGYYKRAETTAGV 480
Qy 481 FDEDGYYRTGDVFAEIAPDRLVYVDRRNNVLKLAQGEFVTLAKLEAVFGNSPLIRQIYVY 540
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 481 FDEDGYYRTGDVFAEIAPDRLVYVDRRNNVLKLAQGEFVTLAKLEAVFGNSPLIRQIYVY 540
Qy 541 GNSAQPYLLAVVVPTEEALASGDPETLKPKIADSLQQVAKEAGLQSYEVPRDFIIETTPF 600
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 541 GNSAQPYLLAVVVPTEEALASGDPETLKPKIADSLQQVAKEAGLQSYEVPRDFIIETTPF 600
Qy 601 SLENGLLTGIRKLAWPKLKQHYGERLEQMYADLAAGQANELAELRRNGAQAPVLQTVSRA 660
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 601 SLENGLLTGIRKLAWPKLKQHYGERLEQMYADLAAGQANELAELRRNGAQAPVLQTVSRA 660
Qy 661 AGAMLGSAASDLSPDAHFTDLGGDSLSALTFGNLLREIFDVDVPVGVIVSPANDLAAIA S 720
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 661 AGAMLGSAASDLSPDAHFTDLGGDSLSALTFGNLLREIFDVDVPVGVIVSPANDLAAIA S 720
Qy 721 YIEAERQGSKRPTFASVHGRDATVVRAADLTLDKFLDAETLAAAPNLPKPATEVRTVLLT 780
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 721 YIEAERQGSKRPTFASVHGRDATVVRAADLTLDKFLDAETLAAAPNLPKPATEVRTVLLT 780
Qy 781 GATGFLGRYLALEWLERMDMVDGKVIALVRARSDEEARARLDKTFDSGDPKLLAHYQQLA 840
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 781 GATGFLGRYLALEWLERMDMVDGKVIALVRARSDEEARARLDKTFDSGDPKLLAHYQQLA 840
Qy 841 ADHLEVIAGDKGEANLGLGQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNALGTAEL 900
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 841 ADHLEVIAGDKGEANLGLGQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNALGTAEL 900
Qy 901 IRLALTSKQKPYTYVSTIGVGDQIEPGKFVENADIRQMSATRAINDSYANGYGNSKWAGE 960
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 901 IRLALTSKQKPYTYVSTIGVGDQIEPGKFVENADIRQMSATRAINDSYANGYGNSKWAGE 960
Qy 961 VLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMFTRLMLSLVATGIAPGSFYELDA 1020
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 961 VLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMFTRLMLSLVATGIAPGSFYELDA 1020
Qy 1021 DGNRQRAHYDGLPVEFIAAAISTLGSQITDSDTGFQTYHVMNPYDDGVGLDEYVDWLVDA 1080
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1021 DGNRQRAHYDGLPVEFIAAAISTLGSQITDSDTGFQTYHVMNPYDDGVGLDEYVDWLVDA 1080
Qy 1081 GYSIERIADYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIAPTDVFRAA 1140
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 1081 GYSIERIADYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIAPTDVFRAA 1140
Qy 1141 VQEAKIGPDKDIPHVSPPVIVKYITDLQLLGLL 1173
|||||||||||||||||||||||||||||||||
Db 1141 VQEAKIGPDKDIPHVSPPVIVKYITDLQLLGLL 1173
RESULT 32
A0A1X0KKW9_MYCSC
ID A0A1X0KKW9_MYCSC Unreviewed; 1177 AA.
AC A0A1X0KKW9;
DT 05-JUL-2017, integrated into UniProtKB/TrEMBL.
DT 05-JUL-2017, sequence version 1.
DT 08-OCT-2025, entry version 33.
DE RecName: Full=Carboxylic acid reductase {ECO:0000256|HAMAP-Rule:MF_02247};
DE Short=CAR {ECO:0000256|HAMAP-Rule:MF_02247};
DE EC=1.2.1.- {ECO:0000256|HAMAP-Rule:MF_02247};
DE AltName: Full=ATP/NADPH-dependent carboxylic acid reductase {ECO:0000256|HAMAP-Rule:MF_02247};
GN Name=car {ECO:0000256|HAMAP-Rule:MF_02247};
GN ORFNames=BST44_03180 {ECO:0000313|EMBL:ORB75922.1};
OS Mycobacterium scrofulaceum.
OC Bacteria; Bacillati; Actinomycetota; Actinomycetes; Mycobacteriales;
OC Mycobacteriaceae; Mycobacterium.
OX NCBI_TaxID=1783 {ECO:0000313|EMBL:ORB75922.1, ECO:0000313|Proteomes:UP000192601};
RN [1] {ECO:0000313|EMBL:ORB75922.1, ECO:0000313|Proteomes:UP000192601}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=DSM 43992 {ECO:0000313|EMBL:ORB75922.1,
RC ECO:0000313|Proteomes:UP000192601};
RA Tortoli E., Trovato A., Cirillo D.M.;
RT "The new phylogeny of genus Mycobacterium.";
RL Submitted (FEB-2017) to the EMBL/GenBank/DDBJ databases.
CC -!- FUNCTION: Catalyzes the ATP- and NADPH-dependent reduction of
CC carboxylic acids to the corresponding aldehydes. {ECO:0000256|HAMAP-
CC Rule:MF_02247}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a carboxylate + ATP + NADPH + H(+) = an aldehyde + AMP +
CC diphosphate + NADP(+); Xref=Rhea:RHEA:50916, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:17478, ChEBI:CHEBI:29067, ChEBI:CHEBI:30616,
CC ChEBI:CHEBI:33019, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:456215; Evidence={ECO:0000256|HAMAP-Rule:MF_02247};
CC -!- COFACTOR:
CC Name=pantetheine 4'-phosphate; Xref=ChEBI:CHEBI:47942;
CC Evidence={ECO:0000256|HAMAP-Rule:MF_02247};
CC Note=Binds 1 phosphopantetheine covalently. {ECO:0000256|HAMAP-
CC Rule:MF_02247};
CC -!- DOMAIN: The N-terminal domain likely catalyzes substrate activation by
CC formation of an initial acyl-AMP intermediate, the central region
CC contains the phosphopantetheine attachment site, and the C-terminal
CC domain catalyzes the reduction by NADPH of the intermediate thioester
CC formed from the attack of the phosphopantetheine thiol at the carbonyl
CC carbon of acyl-AMP. {ECO:0000256|HAMAP-Rule:MF_02247}.
CC -!- SIMILARITY: Belongs to the ATP-dependent AMP-binding enzyme family.
CC Carboxylic acid reductase subfamily. {ECO:0000256|HAMAP-Rule:MF_02247}.
CC -!- CAUTION: Lacks conserved residue(s) required for the propagation of
CC feature annotation. {ECO:0000256|HAMAP-Rule:MF_02247}.
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:ORB75922.1}.
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DR EMBL; MVIJ01000002; ORB75922.1; -; Genomic_DNA.
DR RefSeq; WP_083175072.1; NZ_MVIJ01000002.1.
DR AlphaFoldDB; A0A1X0KKW9; -.
DR STRING; 1783.BST44_03180; -.
DR OrthoDB; 2472181at2; -.
DR Proteomes; UP000192601; Unassembled WGS sequence.
DR GO; GO:0016020; C:membrane; IEA:TreeGrafter.
DR GO; GO:0005524; F:ATP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0004467; F:long-chain fatty acid-CoA ligase activity; IEA:TreeGrafter.
DR GO; GO:0050661; F:NADP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0016620; F:oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor; IEA:UniProtKB-UniRule.
DR GO; GO:0031177; F:phosphopantetheine binding; IEA:UniProtKB-UniRule.
DR CDD; cd17632; AFD_CAR-like; 1.
DR CDD; cd05235; SDR_e1; 1.
DR Gene3D; 1.10.1200.10; ACP-like; 1.
DR Gene3D; 3.40.50.12780; N-terminal domain of ligase-like; 1.
DR Gene3D; 3.40.50.720; NAD(P)-binding Rossmann-like Domain; 1.
DR HAMAP; MF_02247; Carbox_acid_reduct; 1.
DR InterPro; IPR036736; ACP-like_sf.
DR InterPro; IPR020845; AMP-binding_CS.
DR InterPro; IPR000873; AMP-dep_synth/lig_dom.
DR InterPro; IPR042099; ANL_N_sf.
DR InterPro; IPR046407; CAR.
DR InterPro; IPR013120; Far_NAD-bd.
DR InterPro; IPR036291; NAD(P)-bd_dom_sf.
DR InterPro; IPR020806; PKS_PP-bd.
DR InterPro; IPR009081; PP-bd_ACP.
DR InterPro; IPR010080; Thioester_reductase-like_dom.
DR NCBIfam; NF041592; carboxyl_red; 1.
DR NCBIfam; TIGR01746; Thioester-redct; 1.
DR PANTHER; PTHR43272:SF33; AMP-BINDING DOMAIN-CONTAINING PROTEIN-RELATED; 1.
DR PANTHER; PTHR43272; LONG-CHAIN-FATTY-ACID--COA LIGASE; 1.
DR Pfam; PF00501; AMP-binding; 1.
DR Pfam; PF07993; NAD_binding_4; 1.
DR Pfam; PF00550; PP-binding; 1.
DR SMART; SM00823; PKS_PP; 1.
DR SUPFAM; SSF56801; Acetyl-CoA synthetase-like; 1.
DR SUPFAM; SSF47336; ACP-like; 1.
DR SUPFAM; SSF51735; NAD(P)-binding Rossmann-fold domains; 1.
DR PROSITE; PS00455; AMP_BINDING; 1.
DR PROSITE; PS50075; CARRIER; 1.
PE 3: Inferred from homology;
KW ATP-binding {ECO:0000256|ARBA:ARBA00022840, ECO:0000256|HAMAP-
KW Rule:MF_02247}; NADP {ECO:0000256|HAMAP-Rule:MF_02247};
KW Nucleotide-binding {ECO:0000256|ARBA:ARBA00022741, ECO:0000256|HAMAP-
KW Rule:MF_02247}; Oxidoreductase {ECO:0000256|HAMAP-Rule:MF_02247};
KW Phosphopantetheine {ECO:0000256|ARBA:ARBA00022450, ECO:0000256|HAMAP-
KW Rule:MF_02247};
KW Phosphoprotein {ECO:0000256|ARBA:ARBA00022553, ECO:0000256|HAMAP-
KW Rule:MF_02247}; Reference proteome {ECO:0000313|Proteomes:UP000192601}.
FT DOMAIN 648..723
FT /note="Carrier"
FT /evidence="ECO:0000259|PROSITE:PS50075"
FT BINDING 294
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 389
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 410..411
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 415
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 488
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 500..503
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 509
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 609
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 780..783
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 807
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 817
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 851..852
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 877..879
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 917
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 953
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 957
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT MOD_RES 682
FT /note="O-(pantetheine 4'-phosphoryl)serine"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
SQ SEQUENCE 1177 AA; 129222 MW; 72388FE4ADC11BDC CRC64;
Query Match 86.6%; Score 5170; Length 1177;
Best Local Similarity 84.3%;
Matches 996; Conservative 87; Mismatches 86; Indels 12; Gaps 5;
Qy 1 MSTATHDERLDRRVHELIATDPQFAAAQPDPAITAALEQPGLRLPQIIRTVLDGYADRPA 60
||| |||||:||: || | |||||||:||||| ||||:|||||||:|||||||||||||
Db 1 MSTINHDERLERRIEELTANDPQFAAARPDPAIEAALEEPGLRLPQVIRTVLDGYADRPA 60
Qy 61 LGQRVVEFVTDAKTGRTSAQLLPRFETITYSEVAQRVSALGRALSDDAVHPGDRVCVLGF 120
| | |||| |: :||| :|||||||::| |: |||||||| : | | |||||:|||
Db 61 LAHRAVEFVEDSASGRTRLELLPRFETLSYRELGDRVSALGRAWAHDEVRVGDRVCILGF 120
Qy 121 NSVDYATIDMALGAIGAVSVPLQTSAAISSLQPIVAETEPTLIASSVNQLSDAVQLITGA 180
|||||||||||| : ||||||||||:::|| |||||||||:||:| ||| |||:||
Db 121 NSVDYATIDMALATVSAVSVPLQTSASLTSLHPIVAETEPTVIAASANQLPDAVELILSG 180
Qy 181 EQAPTRLVVFDYHPQVDDQREAVQDAAARLSSTGVAVQTLAELLERGKDLPAVAEPPADE 240
: | :||||||||:|||:||||: | ||: || |:||||:||||: || | :||
Db 181 HR-PAKLVVFDYHPEVDDEREAVETARTRLADAGVVVETLAEVLERGRALPDAELPASDE 239
Qy 241 -DSLALLIYTSGSTGAPKGAMYPQSNVGKMWRRGSKNWFGESAASITLNFMPMSHVMGRS 299
| |||||||||||||||||||||||| |:|||||:|||||||||||||||||||||||
Db 240 PDPLALLIYTSGSTGAPKGAMYPQSNVAKIWRRGSRNWFGESAASITLNFMPMSHVMGRG 299
Qy 300 ILYGTLGNGGTAYFAARSDLSTLLEDLELVRPTELNFVPRIWETLYGEFQRQVERRLSEA 359
|||||||||||||||||||||||||||||||||||||||||||||:|||||||||||:
Db 300 ILYGTLGNGGTAYFAARSDLSTLLEDLELVRPTELNFVPRIWETLFGEFQRQVERRLA-- 357
Qy 360 GDAGERRAVEAEVLAEQRQYLLGGRFTFAMTSSAPISPELRNWVESLLEMHLMDGYGSTE 419
| :|:||||||||||||||||||: |||| ||| ||||||||||||:|||||||||||
Db 358 -DGADRQAVEAEVLAEQRQYLLGGRYIFAMTGSAPTSPELRNWVESLLQMHLMDGYGSTE 416
Qy 420 AGMVLFDGEIQRPPVIDYKLVDVPDLGYFSTDRPHPRGELLLRTENMFPGYYKRAETTAG 479
||||||||||||||||||||||||||||| ||||||||||:||||||||||||||| ||
Db 417 AGMVLFDGEIQRPPVIDYKLVDVPDLGYFGTDRPHPRGELVLRTENMFPGYYKRAEITAN 476
Qy 480 VFDEDGYYRTGDVFAEIAPDRLVYVDRRNNVLKLAQGEFVTLAKLEAVFGNSPLIRQIYV 539
||||||||||||||||:|||:|||||||||||||||||||||||||| ||||||:|||||
Db 477 VFDEDGYYRTGDVFAEVAPDKLVYVDRRNNVLKLAQGEFVTLAKLEAEFGNSPLVRQIYV 536
Qy 540 YGNSAQPYLLAVVVPTEEALASGDPETLKPKIADSLQQVAKEAGLQSYEVPRDFIIETTP 599
||||||||||||||||:||| ||| || ||||||| ||::||||||||||||||||||
Db 537 YGNSAQPYLLAVVVPTQEALGRWDPEALKGKIADSLQNVARQAGLQSYEVPRDFIIETTP 596
Qy 600 FSLENGLLTGIRKLAWPKLKQHYGERLEQMYADLAAGQANELAELRRNGAQAPVLQTVSR 659
|||||||||||||||||||||||||||||:||:|| |||||||||||:|| |||||||||
Db 597 FSLENGLLTGIRKLAWPKLKQHYGERLEQLYAELAEGQANELAELRRSGANAPVLQTVSR 656
Qy 660 AAGAMLGSAASDLSPDAHFTDLGGDSLSALTFGNLLREIFDVDVPVGVIVSPANDLAAIA 719
|| ||||:||:||||||||||||||||||||||||||||||:|||||||||||:|| |||
Db 657 AAAAMLGTAATDLSPDAHFTDLGGDSLSALTFGNLLREIFDIDVPVGVIVSPASDLQAIA 716
Qy 720 SYIEAERQGSKRPTFASVHGRDATVVRAADLTLDKFLDAETLAAAPNLPKPATEVRTVLL 779
:||| |||||||||||:||||:|| | |:||||||||:||||||||:|||| ||||||||
Db 717 NYIEGERQGSKRPTFAAVHGREATTVHASDLTLDKFLEAETLAAAPSLPKPTTEVRTVLL 776
Qy 780 TGATGFLGRYLALEWLERMDMVDGKVIALVRARSDEEARARLDKTF----DSGDPKLLAH 835
||||||||||||||||||||:|||||||||||:||:|||||||||| |||||:||
Db 777 TGATGFLGRYLALEWLERMDLVDGKVIALVRAKSDDEARARLDKTFGVGSPQGDPKLVAH 836
Qy 836 YQQLAADHLEVIAGDKGEANLGLGQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNAL 895
|::||||||||||||||| |||| | |||||||||||||||||||||||||||||||||
Db 837 YRELAADHLEVIAGDKGEPNLGLDQQTWQRLADTVDVIVDPAALVNHVLPYSELFGPNAL 896
Qy 896 GTAELIRLALTSKQKPYTYVSTIGVGDQIEPGKFVENADIRQMSATRAINDSYANGYGNS 955
|||||||:|||:| |||||||||||||||:||:|||:|||||:|||||:||:||||||||
Db 897 GTAELIRIALTTKLKPYTYVSTIGVGDQIKPGQFVEDADIRQISATRAVNDNYANGYGNS 956
Qy 956 KWAGEVLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMFTRLMLSLVATGIAPGSF 1015
||||||||||||||||||||||||||||||||||||||||||||||||||||||:|||||
Db 957 KWAGEVLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMFTRLMLSLVATGVAPGSF 1016
Qy 1016 YELDADGNRQRAHYDGLPVEFIAAAISTLGSQITDSDT---GFQTYHVMNPYDDGVGLDE 1072
|||||||||||:||||||||||||||||||:|: |: : ||||||||||||||:||||
Db 1017 YELDADGNRQRSHYDGLPVEFIAAAISTLGTQVLDNASGRDGFQTYHVMNPYDDGIGLDE 1076
Qy 1073 YVDWLVDAGYSIERIADYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIA 1132
|||||:|||| |:||||| ||||||| ::| ||:||||||||||||||: ||||||||:|
Db 1077 YVDWLIDAGYGIQRIADYGEWLRRFEGTMRGLPERQRQYSLLPLLHNYQQPEKPINGSMA 1136
Qy 1133 PTDVFRAAVQEAKIGPDKDIPHVSPPVIVKYITDLQLLGLL 1173
||| ||||||||||||||||||||||:|||| |||||||||
Db 1137 PTDRFRAAVQEAKIGPDKDIPHVSPPIIVKYATDLQLLGLL 1177
RESULT 7
J5E0P6_9MYCO
ID J5E0P6_9MYCO Unreviewed; 1170 AA.
AC J5E0P6;
DT 31-OCT-2012, integrated into UniProtKB/TrEMBL.
DT 31-OCT-2012, sequence version 1.
DT 08-OCT-2025, entry version 62.
DE RecName: Full=Carboxylic acid reductase {ECO:0000256|HAMAP-Rule:MF_02247};
DE Short=CAR {ECO:0000256|HAMAP-Rule:MF_02247};
DE EC=1.2.1.- {ECO:0000256|HAMAP-Rule:MF_02247};
DE AltName: Full=ATP/NADPH-dependent carboxylic acid reductase {ECO:0000256|HAMAP-Rule:MF_02247};
GN Name=car {ECO:0000256|HAMAP-Rule:MF_02247};
GN ORFNames=MCOL_V219786 {ECO:0000313|EMBL:EJO87171.1};
OS Mycobacterium colombiense CECT 3035.
OC Bacteria; Bacillati; Actinomycetota; Actinomycetes; Mycobacteriales;
OC Mycobacteriaceae; Mycobacterium; Mycobacterium avium complex (MAC).
OX NCBI_TaxID=1041522 {ECO:0000313|EMBL:EJO87171.1, ECO:0000313|Proteomes:UP000006455};
RN [1] {ECO:0000313|EMBL:EJO87171.1, ECO:0000313|Proteomes:UP000006455}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=CECT 3035 {ECO:0000313|EMBL:EJO87171.1,
RC ECO:0000313|Proteomes:UP000006455};
RX PubMed=21952541; DOI=10.1128/JB.05928-11;
RA Gonzalez-Perez M., Murcia M.I., Landsman D., Jordan I.K.,
RA Marino-Ramirez L.;
RT "Genome sequence of the Mycobacterium colombiense type strain, CECT 3035.";
RL J. Bacteriol. 193:5866-5867(2011).
CC -!- FUNCTION: Catalyzes the ATP- and NADPH-dependent reduction of
CC carboxylic acids to the corresponding aldehydes. {ECO:0000256|HAMAP-
CC Rule:MF_02247}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=a carboxylate + ATP + NADPH + H(+) = an aldehyde + AMP +
CC diphosphate + NADP(+); Xref=Rhea:RHEA:50916, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:17478, ChEBI:CHEBI:29067, ChEBI:CHEBI:30616,
CC ChEBI:CHEBI:33019, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349,
CC ChEBI:CHEBI:456215; Evidence={ECO:0000256|HAMAP-Rule:MF_02247};
CC -!- COFACTOR:
CC Name=pantetheine 4'-phosphate; Xref=ChEBI:CHEBI:47942;
CC Evidence={ECO:0000256|HAMAP-Rule:MF_02247};
CC Note=Binds 1 phosphopantetheine covalently. {ECO:0000256|HAMAP-
CC Rule:MF_02247};
CC -!- DOMAIN: The N-terminal domain likely catalyzes substrate activation by
CC formation of an initial acyl-AMP intermediate, the central region
CC contains the phosphopantetheine attachment site, and the C-terminal
CC domain catalyzes the reduction by NADPH of the intermediate thioester
CC formed from the attack of the phosphopantetheine thiol at the carbonyl
CC carbon of acyl-AMP. {ECO:0000256|HAMAP-Rule:MF_02247}.
CC -!- SIMILARITY: Belongs to the ATP-dependent AMP-binding enzyme family.
CC Carboxylic acid reductase subfamily. {ECO:0000256|HAMAP-Rule:MF_02247}.
CC -!- CAUTION: Lacks conserved residue(s) required for the propagation of
CC feature annotation. {ECO:0000256|HAMAP-Rule:MF_02247}.
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:EJO87171.1}.
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DR EMBL; AFVW02000006; EJO87171.1; -; Genomic_DNA.
DR RefSeq; WP_007774522.1; NZ_AFVW02000006.1.
DR AlphaFoldDB; J5E0P6; -.
DR STRING; 1041522.GCA_002105755_00493; -.
DR GeneID; 31529323; -.
DR eggNOG; COG1022; Bacteria.
DR eggNOG; COG3320; Bacteria.
DR OrthoDB; 2472181at2; -.
DR Proteomes; UP000006455; Unassembled WGS sequence.
DR GO; GO:0016020; C:membrane; IEA:TreeGrafter.
DR GO; GO:0005524; F:ATP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0004467; F:long-chain fatty acid-CoA ligase activity; IEA:TreeGrafter.
DR GO; GO:0050661; F:NADP binding; IEA:UniProtKB-UniRule.
DR GO; GO:0016620; F:oxidoreductase activity, acting on the aldehyde or oxo group of donors, NAD or NADP as acceptor; IEA:UniProtKB-UniRule.
DR GO; GO:0031177; F:phosphopantetheine binding; IEA:UniProtKB-UniRule.
DR CDD; cd17632; AFD_CAR-like; 1.
DR CDD; cd05235; SDR_e1; 1.
DR Gene3D; 1.10.1200.10; ACP-like; 1.
DR Gene3D; 3.40.50.12780; N-terminal domain of ligase-like; 1.
DR Gene3D; 3.40.50.720; NAD(P)-binding Rossmann-like Domain; 1.
DR HAMAP; MF_02247; Carbox_acid_reduct; 1.
DR InterPro; IPR036736; ACP-like_sf.
DR InterPro; IPR020845; AMP-binding_CS.
DR InterPro; IPR000873; AMP-dep_synth/lig_dom.
DR InterPro; IPR042099; ANL_N_sf.
DR InterPro; IPR046407; CAR.
DR InterPro; IPR013120; Far_NAD-bd.
DR InterPro; IPR036291; NAD(P)-bd_dom_sf.
DR InterPro; IPR020806; PKS_PP-bd.
DR InterPro; IPR009081; PP-bd_ACP.
DR InterPro; IPR010080; Thioester_reductase-like_dom.
DR NCBIfam; NF041592; carboxyl_red; 1.
DR NCBIfam; TIGR01746; Thioester-redct; 1.
DR PANTHER; PTHR43272:SF33; AMP-BINDING DOMAIN-CONTAINING PROTEIN-RELATED; 1.
DR PANTHER; PTHR43272; LONG-CHAIN-FATTY-ACID--COA LIGASE; 1.
DR Pfam; PF00501; AMP-binding; 1.
DR Pfam; PF07993; NAD_binding_4; 1.
DR Pfam; PF00550; PP-binding; 1.
DR SMART; SM00823; PKS_PP; 1.
DR SUPFAM; SSF56801; Acetyl-CoA synthetase-like; 1.
DR SUPFAM; SSF47336; ACP-like; 1.
DR SUPFAM; SSF51735; NAD(P)-binding Rossmann-fold domains; 1.
DR PROSITE; PS00455; AMP_BINDING; 1.
DR PROSITE; PS50075; CARRIER; 1.
PE 3: Inferred from homology;
KW ATP-binding {ECO:0000256|ARBA:ARBA00022840, ECO:0000256|HAMAP-
KW Rule:MF_02247}; NADP {ECO:0000256|HAMAP-Rule:MF_02247};
KW Nucleotide-binding {ECO:0000256|ARBA:ARBA00022741, ECO:0000256|HAMAP-
KW Rule:MF_02247}; Oxidoreductase {ECO:0000256|HAMAP-Rule:MF_02247};
KW Phosphopantetheine {ECO:0000256|ARBA:ARBA00022450, ECO:0000256|HAMAP-
KW Rule:MF_02247};
KW Phosphoprotein {ECO:0000256|ARBA:ARBA00022553, ECO:0000256|HAMAP-
KW Rule:MF_02247}.
FT DOMAIN 648..723
FT /note="Carrier"
FT /evidence="ECO:0000259|PROSITE:PS50075"
FT BINDING 294
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 389
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 410..411
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 415
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 488
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 500..503
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 509
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 609
FT /ligand="AMP"
FT /ligand_id="ChEBI:CHEBI:456215"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 780..783
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 807
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 817
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 847..848
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 873..875
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 913
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 949
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT BINDING 953
FT /ligand="NADP(+)"
FT /ligand_id="ChEBI:CHEBI:58349"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
FT MOD_RES 682
FT /note="O-(pantetheine 4'-phosphoryl)serine"
FT /evidence="ECO:0000256|HAMAP-Rule:MF_02247"
SQ SEQUENCE 1170 AA; 128099 MW; 546AE4D4CA23FD9D CRC64;
Query Match 90.7%; Score 5417.5; Length 1170;
Best Local Similarity 89.6%;
Matches 1051; Conservative 51; Mismatches 68; Indels 3; Gaps 1;
Qy 1 MSTATHDERLDRRVHELIATDPQFAAAQPDPAITAALEQPGLRLPQIIRTVLDGYADRPA 60
|||| ||| ||||: |:|||||||| :|||||||| |||||||||||||||||||||||
Db 1 MSTAIHDEELDRRIEHLVATDPQFAATRPDPAITAATEQPGLRLPQIIRTVLDGYADRPA 60
Qy 61 LGQRVVEFVTDAKTGRTSAQLLPRFETITYSEVAQRVSALGRALSDDAVHPGDRVCVLGF 120
||||||||| |||||||||:|||||||:|| |: ||||||||| : |:| ||||||||||
Db 61 LGQRVVEFVKDAKTGRTSAELLPRFETVTYGELGQRVSALGRAWASDSVSPGDRVCVLGF 120
Qy 121 NSVDYATIDMALGAIGAVSVPLQTSAAISSLQPIVAETEPTLIASSVNQLSDAVQLITGA 180
|||||||||:||| |||||||||||||:||||||||||||:||||||||| |||:||
Db 121 NSVDYATIDIALGTIGAVSVPLQTSAALSSLQPIVAETEPSLIASSVNQLPDAVELILAG 180
Qy 181 EQAPTRLVVFDYHPQVDDQREAVQDAAARLSSTGVAVQTLAELLERGKDLPAVAEPPADE 240
: | ||||||| |:||||||||: | |||: || |: ||::| |||||| | | ||
Db 181 DHVPGRLVVFDYQPEVDDQREAVESAVARLAGIGVVVEQLADVLRRGKDLPPVPEQQTDE 240
Qy 241 DSLALLIYTSGSTGAPKGAMYPQSNVGKMWRRGSKNWFGESAASITLNFMPMSHVMGRSI 300
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||| |
Db 241 DSLALLIYTSGSTGAPKGAMYPQSNVGKMWRRGSKNWFGESAASITLNFMPMSHVMGRGI 300
Qy 301 LYGTLGNGGTAYFAARSDLSTLLEDLELVRPTELNFVPRIWETLYGEFQRQVERRLSEAG 360
|||||||||||||||||||||||||||||||||:||||||||||||||||||||||:
Db 301 LYGTLGNGGTAYFAARSDLSTLLEDLELVRPTEMNFVPRIWETLYGEFQRQVERRLT--- 357
Qy 361 DAGERRAVEAEVLAEQRQYLLGGRFTFAMTSSAPISPELRNWVESLLEMHLMDGYGSTEA 420
| :| ||||||| ||||||||||| |||| ||| ||||: | ||||:||||||||||||
Db 358 DGADREAVEAEVLEEQRQYLLGGRFIFAMTGSAPTSPELKKWAESLLQMHLMDGYGSTEA 417
Qy 421 GMVLFDGEIQRPPVIDYKLVDVPDLGYFSTDRPHPRGELLLRTENMFPGYYKRAETTAGV 480
|||||||||||||||||||||||||||||||||:||||||||||||||||||||||||||
Db 418 GMVLFDGEIQRPPVIDYKLVDVPDLGYFSTDRPYPRGELLLRTENMFPGYYKRAETTAGV 477
Qy 481 FDEDGYYRTGDVFAEIAPDRLVYVDRRNNVLKLAQGEFVTLAKLEAVFGNSPLIRQIYVY 540
||:|||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 478 FDDDGYYRTGDVFAEIAPDRLVYVDRRNNVLKLAQGEFVTLAKLEAVFGNSPLIRQIYVY 537
Qy 541 GNSAQPYLLAVVVPTEEALASGDPETLKPKIADSLQQVAKEAGLQSYEVPRDFIIETTPF 600
|||||||||||||||||||| | | ||||||||||:|||| ||||||||||||||||||
Db 538 GNSAQPYLLAVVVPTEEALADNDIEALKPKIADSLQKVAKETGLQSYEVPRDFIIETTPF 597
Qy 601 SLENGLLTGIRKLAWPKLKQHYGERLEQMYADLAAGQANELAELRRNGAQAPVLQTVSRA 660
:|||||||||||||||||||||||||||||||||||||||||||||:|||||||||||||
Db 598 TLENGLLTGIRKLAWPKLKQHYGERLEQMYADLAAGQANELAELRRSGAQAPVLQTVSRA 657
Qy 661 AGAMLGSAASDLSPDAHFTDLGGDSLSALTFGNLLREIFDVDVPVGVIVSPANDLAAIA S 720
| ||||:| ||| |||||||||||||||||||||||||||||||||||||||||||||:
Db 658 AAAMLGAATGDLSGDAHFTDLGGDSLSALTFGNLLREIFDVDVPVGVIVSPANDLAAIA A 717
Qy 721 YIEAERQGSKRPTFASVHGRDATVVRAADLTLDKFLDAETLAAAPNLPKPATEVRTVLLT 780
|||||||||||||||:|||| || ||| ||||||||| || ||:||||:|||||||||
Db 718 YIEAERQGSKRPTFAAVHGRGATTVRAGDLTLDKFLDEALLAGAPSLPKPSTEVRTVLLT 777
Qy 781 GATGFLGRYLALEWLERMDMVDGKVIALVRARSDEEARARLDKTFDSGDPKLLAHYQQLA 840
||||||||||||:||||||||||||||||||||||||||||||||||||| ||||||:||
Db 778 GATGFLGRYLALDWLERMDMVDGKVIALVRARSDEEARARLDKTFDSGDPTLLAHYQELA 837
Qy 841 ADHLEVIAGDKGEANLGLGQDVWQRLADTVDVIVDPAALVNHVLPYSELFGPNALGTAEL 900
|||||||||||||||||| | |||||||||:||||||||||||||||||||||||||||
Db 838 ADHLEVIAGDKGEANLGLDQQTWQRLADTVDIIVDPAALVNHVLPYSELFGPNALGTAEL 897
Qy 901 IRLALTSKQKPYTYVSTIGVGDQIEPGKFVENADIRQMSATRAINDSYANGYGNSKWAGE 960
||:|||||||||||||||||||||:||||||||||||:|||| |||:|||||||||||||
Db 898 IRIALTSKQKPYTYVSTIGVGDQIQPGKFVENADIRQISATREINDNYANGYGNSKWAGE 957
Qy 961 VLLREAHDLCGLPVAVFRCDMILADTTYAGQLNLPDMFTRLMLSLVATGIAPGSFYELDA 1020
||||||||||||||:|||||||||||||||||||||||||||||:||||||| |||||||
Db 958 VLLREAHDLCGLPVSVFRCDMILADTTYAGQLNLPDMFTRLMLSVVATGIAPRSFYELDA 1017
Qy 1021 DGNRQRAHYDGLPVEFIAAAISTLGSQITDSDTGFQTYHVMNPYDDGVGLDEYVDWLVDA 1080
:||||||||||||||||||||||||:|||||||||||||||||||||:|||||:||||:|
Db 1018 EGNRQRAHYDGLPVEFIAAAISTLGTQITDSDTGFQTYHVMNPYDDGIGLDEYIDWLVEA 1077
Qy 1081 GYSIERIADYSEWLRRFETSLRALPDRQRQYSLLPLLHNYRTPEKPINGSIAPTDVFRAA 1140
||||||| ||||||||||||||||||||||||||||||||: ||||||||:|||||||||
Db 1078 GYSIERIPDYSEWLRRFETSLRALPDRQRQYSLLPLLHNYQKPEKPINGSMAPTDVFRAA 1137
Qy 1141 VQEAKIGPDKDIPHVSPPVIVKYITDLQLLGLL 1173
|||||||||||||||| ||||||||:|||||||
Db 1138 VQEAKIGPDKDIPHVSAPVIVKYITNLQLLGLL 1170
Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA )
Claims 2, 7 and 10 remain rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention. New grounds of rejection are necessitated by amendment.
Claim 2 is indefinite in the recitation of “a 6-aminoacproic acid substrate” and “as compared to a corresponding wild type CAR of SEQ ID NO: 153….as compared to the corresponding wild type CAR of SEQ ID NO: 153….having activity that is at least 20% higher than the activity of the corresponding wild type CAR of SEQ ID NO: 153…” for the following reasons. It is unclear if the term “substrate” next to 6-aminocaproic acid is merely indicating that 6-aminocaproic acid is the substrate used by the CAR to catalyze the desired conversion, or if the term “6-aminocaproic acid substrate” refers to any compound that has a 6-aminocaproic acid moiety attached to it. If the intended limitation is “forming 6-aminocaproate semialdehyde from 6-aminocaproic acid” the claim should be amended accordingly. The terms “a corresponding wild type CAR of SEQ ID NO: 153…” and “the corresponding wild type CAR of SEQ ID NO: 153…” are unclear because one cannot determine if the comparison is being made with the protein of SEQ ID NO: 153, or some unknown/undefined protein which “corresponds” to the polypeptide of SEQ ID NO: 153. Please note that the term “corresponding wild type CAR of SEQ ID NO: 153…” could be interpreted as any protein having some similarity in structure or function with the recited wild type CAR. It should also be noted that even if the claim were to require the comparison to be made with the protein of SEQ ID NO: 153, 154 or 254, the claim would require the comparison to be made with a group of proteins. The basis for comparison is variable, thus making the determination of as to what is encompassed or excluded by the claim impossible. For example, a protein can be encompassed by the claim if the comparison is made with the wild-type CAR of SEQ ID NO: 153 and at the same excluded from the scope of the claim if the comparison is made with the wild type CAR of SEQ ID NO: 254. In addition, the term “activity” with regard to the CAR enzyme is unclear in the absence of a statement indicating the specific activity of the CAR enzyme being compared. An enzyme can have enzymatic activity, binding activity, antibody eliciting activity. If the intended activity refers to the ability to catalyze the conversion of 6-aminoacproic acid to 6-aminocaproate semialdehyde, the claim should be amended accordingly. Correction is required.
Claim 7 is indefinite in the recitation of “…CAR of claim 1, wherein (a) the one or more amino acid alterations comprise at least two….or more of an amino acid sequence of SEQ ID NO: 153, 152 or 254; (b) the one or more positions comprise at least two…or more amino acid positions of SEQ ID NO: 153…” for the following reasons. The term “an amino acid sequence of SEQ ID NO: 153…” encompasses fragments of the sequences of SEQ ID NO: 153, 152 or 254 due to the recitation of “an”. Please note that the term “an amino acid sequence of SEQ ID NO: X” can be interpreted as any fragment within SEQ ID NO: X. Therefore, it is unclear if the one or more alterations are necessarily limited to alterations at positions that correspond to positions in SEQ ID NO: 153, 152 or 254 which are recited in claim 1. In addition, it is unclear if the “one or more positions” recited in part (b) are positions which are different from the positions recited in claim 1, such as position 335, 141, 245, etc. If the intended limitation is “wherein the CAR of claim 1 comprises two or more alterations at two or more positions corresponding to positions in SEQ ID NO: 153 selected from positions 335…”, the claim should be amended accordingly. For examination purposes, it will be assumed that claim 7 is directed to the protein of claim 1, wherein said protein requires additional alterations at positions that correspond to positions in SEQ ID NO: 153 that may or may not be those recited in claim 1. Correction is required.
Claim 10 is indefinite in the recitation of “the one or more amino acid alterations are alterations from a substitution with an E, D…..or F and combinations thereof of SEQ ID NO: 153, 152 or 254.; (b) the engineered CAR comprises one or more amino acid alterations selected from the group consisting of N335D…..for the following reasons. The term “combinations thereof of SEQ ID NO: 153, 152 or 254” imply a combination of sequences, wherein said sequences are SEQ ID NO: 153, 152, or 254. The term “alterations selected from a substitution with an E, D…..or F” implies that the alterations to select from are substitutions with any one of the recited amino acids. Therefore, it is unclear as to how an alteration can be a combination of amino acid sequences. Even if one were to interpret the term “combination thereof” as referring to a combination of any of the amino acids recited (e.g., E, D, P….), it is unclear if the substitution is the replacement of one amino acid with a single amino acid, or if the substitution is the replacement of one amino acid with several amino acids, which are a combination of any of the amino acids recited (e.g., replacement of one amino acid with the peptide EDPV). Moreover, the recitation of substitutions such as those in part (b) (e.g., N335D) is meaningless in the absence of the sequence identifier associated with the numerical position where the substitution is present. For examination purposes, it will be assumed that claim 10 is directed to the CAR of claim 1, wherein the amino acid substitutions are made with an E, D, P….or F. Correction is required.
When amending the claims, applicant is advised to carefully review all examined claims and make the necessary changes to ensure proper antecedent basis and dependency.
Claim Rejections - 35 USC § 112(a) or First Paragraph (pre-AIA )
Claims 1-2, 7 and 10 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
This rejection has been discussed at length in the prior Office action. It is maintained for the reasons of record and those set forth below.
Applicant argues that a representative number of species and common structural features has been provided so that one of skill in the art could visualize and/or recognize the members of the genus for each independent claim. Applicant cites case law in support of the argument that adequate written description does not require a perfect correspondence between the members of the genus and the asserted common structural feature for a functionally defined genus. Applicant refers to Examples 4 and 5 where it is asserted that 115 CAR homologs and variants having activity on 6-aminocaproic acid have been provided.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection. The Examiner acknowledges the teachings of the specification, Examples 4 and 5, as well as the cited case law. However, the Examiner disagrees with Applicant’s contention that the entire genus of claimed proteins is adequately described.
Claims 1-2, 7 and 10 as interpreted are directed in part to a genus of variants of the polypeptide of SEQ ID NO: 153 that have carboxylic acid reductase (CAR) activity, wherein said variants have at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, wherein said variants comprise a substitution that correspond to the substitution N335D in the polypeptide of SEQ ID NO: 153. See Claim Rejections - 35 USC § 112(b) or Second Paragraph (pre-AIA ) for claim interpretation.
The claims encompass a large genus of proteins which are substantially unrelated. A polypeptide having at least 85% sequence identity with the polypeptide of SEQ ID NO: 153 allows for any combination of 176 amino acid modifications within SEQ ID NO: 153 (176 = 0.15x1173; SEQ ID NO: 153 has 1173 amino acids). The total number of variants of a polypeptide having a specific number of amino acid substitutions can be calculated from the formula N!x19A/(N-A)!/A!, where N is the length in amino acids of the reference polypeptide and A is the number of allowed substitutions. Thus, the total number of variants having at least 85% sequence identity to the polypeptide of SEQ ID NO: 153 that result from amino acid substitutions is 1173!x19176/(1173-176)!/176! or 8.93x10438 variants. This number is even larger if one takes into accounts variants that result from deletions or insertions.
While it is agreed that the specification discloses a few CAR enzymes which have carboxylic acid reductase activity on 6-aminocaproic acid and a few variants of the protein of SEQ ID NO: 153 that have carboxylic acid reductase activity on 6-aminocaproic acid, wherein said variants comprise all of SEQ ID NO: 153 except for up to 7 substitutions, neither the specification nor the prior art disclose the structural features required in any CAR enzyme that can use 6-aminocaproic acid as a substrate, or the structural features within the polypeptide of SEQ ID NO: 153 that are essential to observe the desired enzymatic activity. No structure/function correlation has been provided which would allow one of skill in the art to determine a priori from an essentially infinite number of variants of the polypeptide of SEQ ID NO: 153 that meet the required % sequence identity limitation, which ones have the desired enzymatic activity. Even if the argument is made that the variants disclosed are representative of all of the members of the genus of proteins encompassed by the claims, it is noted that the art teaches several examples of how even highly structurally homologous polypeptides can have different enzymatic activities. See the teachings of Witkowski et al., Tang et al. and Seffernick et al. previously discussed. Since minor structural differences may result in changes affecting function, and no additional information correlating structure with the desired functional characteristics has been provided, one could have not reasonably concluded that the species disclosed are representative of the structure of all the CAR proteins required by the claims. Therefore, contrary to Applicant’s assertions, one of skill in the art cannot reasonably conclude that the entire genus of CAR enzymes claimed is adequately described by the teachings of the specification and/or the prior art.
Claims 1-2, 7 and 10 remain rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a variant of the polypeptide of SEQ ID NO: 153 that has carboxylic acid reductase activity, wherein said variant comprises all of SEQ ID NO: 153 except for a substitution at the position corresponding to position 335 of the polypeptide of SEQ ID NO: 153, does not reasonably provide enablement for a variant of the polypeptide of SEQ ID NO: 153 having 85% sequence identity to the polypeptide of SEQ ID NO: 153, wherein said variant comprises a substitution at the position corresponding to position 335 of the polypeptide of SEQ ID NO: 153, wherein said variant has carboxylic acid reductase activity. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
This rejection has been discussed at length in the prior Office action. It is maintained for the reasons of record and those set forth below.
Applicant states that the specification provides sufficient description and guidance such that one of skilled in the art could make and use the claimed CAR enzymes in their full scope. Applicant states that the specification teaches numerous CAR homologs including the CAR homologs in SEQ ID NO: 153, 152 and 254 that can serve as parental enzymes from which the recited engineered CAR enzymes can be mutated. Applicant states that the specification discloses specific amino acid positions to be altered and recombinant techniques for mutagenesis, construction and expression of mutated oligonucleotides. According to applicant, it is only a matter of routine experimentation to determine that the protein exhibits carboxylic acid activity. Applicant concludes that one of skill in the art can make and use the claimed enzymes using routine methods and that the claimed enzymes are fully enabled.
Applicant’s arguments have been fully considered but not deemed persuasive to overcome the instant rejection. The Examiner acknowledges the teachings of the specification. However, the Examiner disagrees with Applicant’s contention that the entire scope of the claims is fully enabled by the teachings of the prior art and/or the specification.
With regard to the arguments that the specification discloses specific amino acid positions to be altered and that recombinant techniques for mutagenesis, construction and expression of mutated oligonucleotides are known in the art, it is noted that (i) the claims are not limited solely to variants that comprise all of SEQ ID NO: 153 except for substitutions at the specific amino acid positions within SEQ ID NO: 153 recited in the claims, or the variants disclosed in Table 9, and (ii) the experimentation required to enable the entire scope of the claims is not routine even if mutagenesis methods, construction of mutated polynucleotides, and expression of mutated polynucleotides in host cells are known in the art.
As explained above, the claims require proteins having at least 85% sequence identity to the polypeptide of SEQ ID NO: 153. This amounts to up to 176 amino acid modifications within the polypeptide of SEQ ID NO: 153 The total number of variants having at least 85% sequence identity to the polypeptide of SEQ ID NO: 153 that result from amino acid substitutions is 1173!x19176/(1173-176)!/176! or 8.93x10438 variants. This number is even larger if one takes into accounts variants that result from deletions or insertions. It is reiterated herein that neither the specification nor the prior art disclose the structural features required in any CAR enzyme that can use 6-aminocaproic acid as a substrate, or the structural features within the polypeptide of SEQ ID NO: 153 that are essential to observe the desired enzymatic activity. No structure/function correlation has been provided which would allow one of skill in the art to determine from an essentially infinite number of variants of the polypeptide of SEQ ID NO: 153 that meet the required % sequence identity limitation, which ones have the desired enzymatic activity.
While the argument can be made that one could determine which of these infinite number of variants have the desired enzymatic activity by structural homology, the art clearly teaches that (i) there is a high level of unpredictability associated with accurate functional annotation of proteins based solely on structural homology, and (ii) modification of a protein’s amino acid sequence to obtain the desired activity without any guidance/knowledge as to which amino acids in a protein are tolerant of modification and which ones are conserved is highly unpredictable. See the teachings of Singh et al. and Sadowski et al. previously discussed.
While it is agreed that methods of generating or isolating variants of a protein as well as enzymatic assays were known in the art at the time of the invention, it was not routine in the art to screen by a trial and error process for an essentially infinite number of proteins to find those that have the desired enzymatic activity. In the absence of (i) a rational and predictable scheme for selecting those proteins most likely to have the desired functional features, and/or (ii) a correlation between structure and CAR activity, one of skill in the art would have to test an essentially infinite number of variants of the polypeptide of SEQ ID NO: 153 that meet the recited % sequence identity to determine which ones have the desired enzymatic activity. This is not deemed routine experimentation. Thus, contrary to Applicant’s assertions, one cannot reasonably conclude that the entire genus of enzymes claimed is fully enabled by the teachings of the specification.
Claim Rejections - 35 USC § 102 (AIA )
Claims 1-2, 7 and 10 were rejected under 35 U.S.C. 102(a)(1) as being anticipated by Tarcisio et al. (GenBank accession No. ORW87810 4/20/2017).
In view of Applicant’s amendment of claim 1, which now requires the carboxylic acid reductase to comprise at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, and the fact that the CAR enzyme of Tarcisio et al. does not meet the recited % sequence identity, this rejection is hereby withdrawn.
Claims 1-2, 7 and 10 were rejected under 35 U.S.C. 102(a)(1) as being anticipated by Amande et al. (GenBank accession No. TQR86954 7/10/2019).
In view of Applicant’s amendment of claim 1, which now requires the carboxylic acid reductase to comprise at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, and the fact that the CAR enzyme of Amande et al. does not meet the recited % sequence identity, this rejection is hereby withdrawn.
Claims 1-2, and 10 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Li et al. (GenBank accession No. AAS03357 1/30/2004). This rejection is necessitated by amendment.
Claims 1-2, and 10 are directed in part to a protein which is a variant of the polypeptide of SEQ ID NO: 153 having at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, wherein said variant has carboxylic acid reductase activity, wherein said variant comprises a substitution at the position corresponding to position 391 of the polypeptide of SEQ ID NO: 153, wherein said substitution is with a glycine. See Claim Rejections under 35 USC § 112(b) or Second Paragraph (pre-AIA ) below for claim interpretation.
Li et al. teach a carboxylic acid reductase (CAR) that comprises a substitution at a position corresponding to position 391 of the polypeptide of SEQ ID NO: 153, wherein said substitution is with a glycine. The CAR of Li et al. comprises 99.9% sequence identity to the polypeptide of SEQ ID NO: 153 (99.9% = 1172x100/1173; SEQ ID NO: 153 has 1173 amino acids). See alignment above. In the absence of any additional distinguishing feature not naturally found in a protein, the term “engineered” is a product-by-process limitation which does not convey any structural and/or functional differences between a man-made and a naturally-occurring product. With regard to the specific functional characteristics recited in claim 2, it is noted that the specification asserts that a variant of the polypeptide of SEQ ID NO: 153 having the recited substitution would have those functional characteristics. Therefore, it follows that the CAR enzyme of Li et al. also comprises those functional characteristics. As such, the protein of Li et al. anticipate the instant claims as written/interpreted.
Claims 1-2, 7 and 10 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Tortoli et al. (GenBank accession No. ORB75922 4/11/2017). This rejection is necessitated by amendment.
Claims 1-2, 7 and 10 are directed in part to a protein which is a variant of the polypeptide of SEQ ID NO: 153 having at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, wherein said variant has carboxylic acid reductase activity, wherein said variant comprises substitutions at the positions corresponding to positions 299 and 391 of the polypeptide of SEQ ID NO: 153, wherein said substitutions are with a glycine, and wherein said variant further comprises substitutions at positions corresponding to positions 275 and 812 of the polypeptide of SEQ ID NO: 153. See Claim Rejections under 35 USC § 112(b) or Second Paragraph (pre-AIA ) below for claim interpretation.
Tortoli et al. teach a carboxylic acid reductase (CAR) that comprises substitutions at positions corresponding to positions 299 and 391 of the polypeptide of SEQ ID NO: 153, wherein said substitutions are with a glycine, and further comprises substitutions at positions 275 and 812 of the polypeptide of SEQ ID NO: 153. The CAR of Tortoli et al. comprises 85% sequence identity to the polypeptide of SEQ ID NO: 153 (85% = 996x100/1173; SEQ ID NO: 153 has 1173 amino acids). See alignment above. In the absence of any additional distinguishing feature not naturally found in a protein, the term “engineered” is a product-by-process limitation which does not convey any structural and/or functional differences between a man-made and a naturally-occurring product. With regard to the specific functional characteristics recited in claim 2, it is noted that the specification asserts that a variant of the polypeptide of SEQ ID NO: 153 having the recited substitution would have those functional characteristics. Therefore, it follows that the CAR enzyme of Tortoli et al. also comprises those functional characteristics. As such, the protein of Tortoli et al. anticipate the instant claims as written/interpreted.
Claims 1-2, 7 and 10 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Gonzalez-Perez et al. (GenBank accession No. EJO87171 8/13/2012). This rejection is necessitated by amendment.
Claims 1-2, 7 and 10 are directed in part to a protein which is a variant of the polypeptide of SEQ ID NO: 153 having at least 85% sequence identity to the polypeptide of SEQ ID NO: 153, wherein said variant has carboxylic acid reductase activity, wherein said variant comprises substitutions at the positions corresponding to positions 299 and 391 of the polypeptide of SEQ ID NO: 153, wherein said substitutions are with a glycine. See Claim Rejections under 35 USC § 112(b) or Second Paragraph (pre-AIA ) below for claim interpretation.
Gonzalez-Perez et al. teach a carboxylic acid reductase (CAR) that comprises substitutions at positions corresponding to positions 299 and 391 of the polypeptide of SEQ ID NO: 153, wherein said substitutions are with a glycine. The CAR of Gonzalez-Perez et al. comprises 90% sequence identity to the polypeptide of SEQ ID NO: 153 (90% = 1051x100/1173; SEQ ID NO: 153 has 1173 amino acids). See alignment above. In the absence of any additional distinguishing feature not naturally found in a protein, the term “engineered” is a product-by-process limitation which does not convey any structural and/or functional differences between a man-made and a naturally-occurring product. With regard to the specific functional characteristics recited in claim 2, it is noted that the specification asserts that a variant of the polypeptide of SEQ ID NO: 153 having the recited substitution would have those functional characteristics. Therefore, it follows that the CAR enzyme of Gonzalez-Perez et al. also comprises those functional characteristics. As such, the protein of Gonzalez-Perez et al. anticipate the instant claims as written/interpreted.
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.
Conclusion
No claim is in condition for allowance.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a). A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
Applicant is advised that any Internet email communication by the Examiner has to be authorized by Applicant in written form. See MPEP § 502.03 (II). Without a written authorization by Applicant in place, the USPTO will not respond via Internet email to any Internet correspondence which contains information subject to the confidentiality requirement as set forth in 35 U.S.C. 122. Sample written authorization language can be found in MPEP § 502.03 (II). An Authorization for Internet Communications in a Patent Application or Request to Withdraw Authorization for Internet Communications form (SB/439) can be found at https://www.uspto.gov/patent/forms/ forms-patent-applications-filed-or-after-september-16-2012, which can be electronically filed.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to DELIA M RAMIREZ, Ph.D., whose telephone number is (571) 272-0938. The examiner can normally be reached on Monday-Friday from 8:30 AM to 5:00 PM. If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Robert B. Mondesi, can be reached at (408) 918-7584. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
/DELIA M RAMIREZ/Primary Examiner, Art Unit 1652
DR
May 25, 2026
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