C71DK_TOBAC
ID C71DK_TOBAC Reviewed; 504 AA.
AC Q94FM7;
DT 31-MAY-2011, integrated into UniProtKB/Swiss-Prot.
DT 25-OCT-2005, sequence version 2.
DT 03-AUG-2022, entry version 91.
DE RecName: Full=5-epiaristolochene 1,3-dihydroxylase;
DE Short=NtEAH;
DE EC=1.14.14.149 {ECO:0000269|PubMed:12504906, ECO:0000269|PubMed:15522862, ECO:0000269|PubMed:17715131};
DE AltName: Full=Cytochrome P450 71D20;
GN Name=CYP71D20;
OS Nicotiana tabacum (Common tobacco).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC asterids; lamiids; Solanales; Solanaceae; Nicotianoideae; Nicotianeae;
OC Nicotiana.
OX NCBI_TaxID=4097;
RN [1]
RP NUCLEOTIDE SEQUENCE [MRNA], FUNCTION, INDUCTION BY ELICITOR, AND ACTIVITY
RP REGULATION.
RX PubMed=11556809; DOI=10.1006/abbi.2001.2483;
RA Ralston L., Kwon S.T., Schoenbeck M., Ralston J., Schenk D.J., Coates R.M.,
RA Chappell J.;
RT "Cloning, heterologous expression, and functional characterization of 5-
RT epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum).";
RL Arch. Biochem. Biophys. 393:222-235(2001).
RN [2]
RP SEQUENCE REVISION.
RA Ralston L., Kwon S.T., Schoenbeck M., Ralston J.L., Schenk D.J.,
RA Coates R.M., Chappell J.;
RL Submitted (SEP-2005) to the EMBL/GenBank/DDBJ databases.
RN [3]
RP CATALYTIC ACTIVITY.
RX PubMed=12504906; DOI=10.1016/s0003-9861(02)00613-6;
RA Greenhagen B.T., Griggs P., Takahashi S., Ralston L., Chappell J.;
RT "Probing sesquiterpene hydroxylase activities in a coupled assay with
RT terpene synthases.";
RL Arch. Biochem. Biophys. 409:385-394(2003).
RN [4]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, AND
RP MUTAGENESIS OF SER-368 AND ILE-486.
RX PubMed=15522862; DOI=10.1074/jbc.m411870200;
RA Takahashi S., Zhao Y., O'Maille P.E., Greenhagen B.T., Noel J.P.,
RA Coates R.M., Chappell J.;
RT "Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a
RT cytochrome P450 enzyme catalyzing successive hydroxylations of
RT sesquiterpenes.";
RL J. Biol. Chem. 280:3686-3696(2005).
RN [5]
RP CATALYTIC ACTIVITY, AND MUTAGENESIS OF SER-482; ILE-484 AND ILE-486.
RX PubMed=17715131; DOI=10.1074/jbc.m703378200;
RA Takahashi S., Yeo Y.S., Zhao Y., O'Maille P.E., Greenhagen B.T., Noel J.P.,
RA Coates R.M., Chappell J.;
RT "Functional characterization of premnaspirodiene oxygenase, a cytochrome
RT P450 catalyzing regio- and stereo-specific hydroxylations of diverse
RT sesquiterpene substrates.";
RL J. Biol. Chem. 282:31744-31754(2007).
CC -!- FUNCTION: Involved in the biosynthesis of capsidiol. Catalyzes the
CC successive and independent hydroxylations at the C1 and C3 positions of
CC 5-epiaristolochene. The second hydroxylation step is 8-fold more
CC efficient than the first hydroxylation reaction. Capable of utilizing
CC premnaspirodiene as a substrate. {ECO:0000269|PubMed:11556809,
CC ECO:0000269|PubMed:15522862}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=(+)-5-epi-aristolochene + 2 O2 + 2 reduced [NADPH--hemoprotein
CC reductase] = capsidiol + 2 H(+) + 2 H2O + 2 oxidized [NADPH--
CC hemoprotein reductase]; Xref=Rhea:RHEA:28226, Rhea:RHEA-COMP:11964,
CC Rhea:RHEA-COMP:11965, ChEBI:CHEBI:15377, ChEBI:CHEBI:15378,
CC ChEBI:CHEBI:15379, ChEBI:CHEBI:23925, ChEBI:CHEBI:28283,
CC ChEBI:CHEBI:57618, ChEBI:CHEBI:58210; EC=1.14.14.149;
CC Evidence={ECO:0000269|PubMed:12504906, ECO:0000269|PubMed:15522862,
CC ECO:0000269|PubMed:17715131};
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413;
CC -!- ACTIVITY REGULATION: Inhibited by ancymidol and ketoconazole.
CC {ECO:0000269|PubMed:11556809}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=19.18 uM for 5-epiaristolochene {ECO:0000269|PubMed:15522862};
CC KM=6.38 uM for 1-deoxycapsidiol {ECO:0000269|PubMed:15522862};
CC KM=1.74 uM for 3-deoxycapsidiol {ECO:0000269|PubMed:15522862};
CC pH dependence:
CC Optimum pH is 7.5. {ECO:0000269|PubMed:15522862};
CC -!- SUBCELLULAR LOCATION: Membrane {ECO:0000305}; Single-pass membrane
CC protein {ECO:0000305}.
CC -!- INDUCTION: Up-regulated 6 to 9 hours after elicitor-treatment and then
CC declines by 12 hours. {ECO:0000269|PubMed:11556809}.
CC -!- SIMILARITY: Belongs to the cytochrome P450 family. {ECO:0000305}.
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DR EMBL; AF368376; AAK62342.2; -; mRNA.
DR RefSeq; NP_001311564.1; NM_001324635.1.
DR AlphaFoldDB; Q94FM7; -.
DR SMR; Q94FM7; -.
DR GeneID; 107759261; -.
DR KEGG; ag:AAK62342; -.
DR KEGG; nta:107759261; -.
DR BioCyc; MetaCyc:EAH-MON; -.
DR BRENDA; 1.14.14.149; 3645.
DR SABIO-RK; Q94FM7; -.
DR Proteomes; UP000084051; Unplaced.
DR GO; GO:0016021; C:integral component of membrane; IEA:UniProtKB-KW.
DR GO; GO:0102170; F:5-epi-aristolochene-1,3-dihydroxylase activity; IEA:UniProtKB-EC.
DR GO; GO:0020037; F:heme binding; IEA:InterPro.
DR GO; GO:0005506; F:iron ion binding; IEA:InterPro.
DR Gene3D; 1.10.630.10; -; 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 Pfam; PF00067; p450; 1.
DR PRINTS; PR00463; EP450I.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; SSF48264; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 1: Evidence at protein level;
KW Heme; Iron; Membrane; Metal-binding; Monooxygenase; Oxidoreductase;
KW Reference proteome; Transmembrane; Transmembrane helix.
FT CHAIN 1..504
FT /note="5-epiaristolochene 1,3-dihydroxylase"
FT /id="PRO_0000409470"
FT TRANSMEM 2..22
FT /note="Helical"
FT /evidence="ECO:0000255"
FT BINDING 442
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:0000250"
FT MUTAGEN 368
FT /note="S->A,T: Decreased substrate affinity but catalytic
FT activities unchanged."
FT /evidence="ECO:0000269|PubMed:15522862"
FT MUTAGEN 368
FT /note="S->C,V: Decreased synthesis of 3-deoxycapsidiol and
FT loss of production of capsidiol."
FT /evidence="ECO:0000269|PubMed:15522862"
FT MUTAGEN 368
FT /note="S->I,F: Loss of both catalytic activities."
FT /evidence="ECO:0000269|PubMed:15522862"
FT MUTAGEN 482
FT /note="S->V: Loss of activity toward premnaspirodiene."
FT /evidence="ECO:0000269|PubMed:17715131"
FT MUTAGEN 484
FT /note="I->V: Loss of activity toward premnaspirodiene."
FT /evidence="ECO:0000269|PubMed:17715131"
FT MUTAGEN 486
FT /note="I->A: Decreased synthesis of 3-deoxycapsidiol and
FT near loss of production of capsidiol. Loss of activity
FT toward premnaspirodiene."
FT /evidence="ECO:0000269|PubMed:15522862,
FT ECO:0000269|PubMed:17715131"
SQ SEQUENCE 504 AA; 57083 MW; 0E5B01665C0D3D44 CRC64;
MQFFSLVSIF LFLSFLFLLR KWKNSNSQSK KLPPGPWKIP ILGSMLHMIG GEPHHVLRDL
AKKYGPLMHL QLGEISAVVV TSRDMAKEVL KTHDVVFASR PKIVAMDIIC YNQSDIAFSP
YGDHWRQMRK ICVMELLNAK NVRSFSSIRR DEVVRLIDSI RSDSSSGELV NFTQRIIWFA
SSMTCRSAFG QVLKGQDIFA KKIREVIGLA EGFDVVDIFP TYKFLHVLSG MKRKLLNAHL
KVDAIVEDVI NEHKKNLAAG KSNGALGGED LIDVLLRLMN DTSLQFPITN DNIKAVIVDM
FAAGTETSST TTVWAMAEMM KNPSVFTKAQ AEVREAFRDK VSFDENDVEE LKYLKLVIKE
TLRLHPPSPL LVPRECREDT DINGYTIPAK TKVMVNVWAL GRDPKYWDDA ESFKPERFEQ
CSVDFFGNNF EFLPFGGGRR ICPGMSFGLA NLYLPLAQLL YHFDWKLPTG IMPRDLDLTE
LSGITIARKG GLYLNATPYQ PSRE
