DOXA_STRS5
ID DOXA_STRS5 Reviewed; 422 AA.
AC Q59971;
DT 19-MAR-2014, integrated into UniProtKB/Swiss-Prot.
DT 01-NOV-1996, sequence version 1.
DT 03-AUG-2022, entry version 85.
DE RecName: Full=Cytochrome P-450 monooxygenase DoxA;
DE EC=1.14.13.181 {ECO:0000269|PubMed:11463295, ECO:0000269|PubMed:8655530, ECO:0000269|PubMed:9098063, ECO:0000269|PubMed:9864343};
DE AltName: Full=13-deoxycarminomycin C-13 hydroxylase;
DE AltName: Full=13-deoxydaunorubicin C-13 hydroxylase;
DE AltName: Full=13-dihydrocarminomycin C-13 hydroxylase;
DE AltName: Full=13-dihydrodaunorubicin C-13 hydroxylase;
GN Name=doxA;
OS Streptomyces sp. (strain C5).
OC Bacteria; Actinobacteria; Streptomycetales; Streptomycetaceae;
OC Streptomyces.
OX NCBI_TaxID=45212;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], FUNCTION, CATALYTIC ACTIVITY, SUBSTRATE
RP SPECIFICITY, AND NOMENCLATURE.
RC STRAIN=C5;
RX PubMed=8655530; DOI=10.1128/jb.178.11.3389-3395.1996;
RA Dickens M.L., Strohl W.R.;
RT "Isolation and characterization of a gene from Streptomyces sp. strain C5
RT that confers the ability to convert daunomycin to doxorubicin on
RT Streptomyces lividans TK24.";
RL J. Bacteriol. 178:3389-3395(1996).
RN [2]
RP FUNCTION, CATALYTIC ACTIVITY, AND SUBSTRATE SPECIFICITY.
RC STRAIN=C5;
RX PubMed=9098063; DOI=10.1128/jb.179.8.2641-2650.1997;
RA Dickens M.L., Priestley N.D., Strohl W.R.;
RT "In vivo and in vitro bioconversion of epsilon-rhodomycinone glycoside to
RT doxorubicin: functions of DauP, DauK, and DoxA.";
RL J. Bacteriol. 179:2641-2650(1997).
RN [3]
RP FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, ACTIVITY
RP REGULATION, SUBSTRATE SPECIFICITY, AND SUBUNIT.
RC STRAIN=C5;
RX PubMed=9864343; DOI=10.1128/jb.181.1.298-304.1999;
RA Walczak R.J., Dickens M.L., Priestley N.D., Strohl W.R.;
RT "Purification, properties, and characterization of recombinant Streptomyces
RT sp. strain C5 DoxA, a cytochrome P-450 catalyzing multiple steps in
RT doxorubicin biosynthesis.";
RL J. Bacteriol. 181:298-304(1999).
RN [4]
RP FUNCTION, CATALYTIC ACTIVITY, AND SUBSTRATE SPECIFICITY.
RC STRAIN=C5;
RX PubMed=11463295; DOI=10.1021/ol015998x;
RA Walczak R.J., Hines J.V., Strohl W.R., Priestley N.D.;
RT "Bioconversion of the anthracycline analogue desacetyladriamycin by
RT recombinant DoxA, a P450-monooxygenase from Streptomyces sp. strain C5.";
RL Org. Lett. 3:2277-2279(2001).
CC -!- FUNCTION: Involved in the biosynthesis of the anthracyclines
CC carminomycin and daunorubicin (daunomycin) which are aromatic
CC polyketide antibiotics that exhibit high cytotoxicity and are widely
CC applied in the chemotherapy of a variety of cancers. In vivo, DoxA
CC catalyzes the C-13 hydroxylation of 13-deoxycarminomycin and 13-
CC deoxydaunorubicin to yield 13-dihydrocarminomycin and 13-
CC dihydrodaunorubicin, respectively, as well as the oxidation of these
CC 13-dihydro-anthracyclines to their respective 13-keto forms,
CC carminomycin and daunorubicin. In vitro, it also catalyzes the C-14
CC hydroxylation of daunorubicin to form doxorubicin (adriamycin),
CC although this strain is not a doxorubicin producer. It is not able to
CC accept anthracyclinones (aglycones) and anthracyclines with a 10-
CC carbomethoxyl moiety. 13-oxidation of the anthracyclines possessing the
CC 4-methoxy substitution is greatly favored. The anthracycline analog
CC desacetyladriamycin can be oxidized to 10-hydroxydesacetyladriamycin.
CC It can only use NADP. DoxA acts jointly with DauV.
CC {ECO:0000269|PubMed:11463295, ECO:0000269|PubMed:8655530,
CC ECO:0000269|PubMed:9098063, ECO:0000269|PubMed:9864343}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=13-deoxydaunorubicin + H(+) + NADPH + O2 = 13-
CC dihydrodaunorubicin + H2O + NADP(+); Xref=Rhea:RHEA:37851,
CC ChEBI:CHEBI:15377, ChEBI:CHEBI:15378, ChEBI:CHEBI:15379,
CC ChEBI:CHEBI:57783, ChEBI:CHEBI:58349, ChEBI:CHEBI:75296,
CC ChEBI:CHEBI:75297; EC=1.14.13.181;
CC Evidence={ECO:0000269|PubMed:11463295, ECO:0000269|PubMed:8655530,
CC ECO:0000269|PubMed:9098063, ECO:0000269|PubMed:9864343};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=13-dihydrodaunorubicin + H(+) + NADPH + O2 = daunorubicin + 2
CC H2O + NADP(+); Xref=Rhea:RHEA:37847, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:15379, ChEBI:CHEBI:57783,
CC ChEBI:CHEBI:58349, ChEBI:CHEBI:64677, ChEBI:CHEBI:75296;
CC EC=1.14.13.181; Evidence={ECO:0000269|PubMed:11463295,
CC ECO:0000269|PubMed:8655530, ECO:0000269|PubMed:9098063,
CC ECO:0000269|PubMed:9864343};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=13-deoxycarminomycin + H(+) + NADPH + O2 = 13-
CC dihydrocarminomycin + H2O + NADP(+); Xref=Rhea:RHEA:56360,
CC ChEBI:CHEBI:15377, ChEBI:CHEBI:15378, ChEBI:CHEBI:15379,
CC ChEBI:CHEBI:57783, ChEBI:CHEBI:58349, ChEBI:CHEBI:140329,
CC ChEBI:CHEBI:140330; Evidence={ECO:0000269|PubMed:11463295,
CC ECO:0000269|PubMed:8655530, ECO:0000269|PubMed:9098063,
CC ECO:0000269|PubMed:9864343};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=13-dihydrocarminomycin + H(+) + NADPH + O2 = carminomycin + 2
CC H2O + NADP(+); Xref=Rhea:RHEA:56364, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:15379, ChEBI:CHEBI:57783,
CC ChEBI:CHEBI:58349, ChEBI:CHEBI:75730, ChEBI:CHEBI:140330;
CC Evidence={ECO:0000269|PubMed:11463295, ECO:0000269|PubMed:8655530,
CC ECO:0000269|PubMed:9098063, ECO:0000269|PubMed:9864343};
CC -!- COFACTOR:
CC Name=heme; Xref=ChEBI:CHEBI:30413; Evidence={ECO:0000250};
CC -!- ACTIVITY REGULATION: Strongly inhibited by dithiothreitol and high
CC ionic strength buffers. {ECO:0000269|PubMed:9864343}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=0.9 uM for daunorubicin (at pH 7.5 and 30 degrees Celsius)
CC {ECO:0000269|PubMed:9864343};
CC KM=1.1 uM for 13-deoxydaunorubicin (at pH 7.5 and 30 degrees Celsius)
CC {ECO:0000269|PubMed:9864343};
CC KM=2.5 uM for 13-dihydrocarminomycin (at pH 7.5 and 30 degrees
CC Celsius) {ECO:0000269|PubMed:9864343};
CC KM=4.6 uM for 13-dihydrodaunorubicin (at pH 7.5 and 30 degrees
CC Celsius) {ECO:0000269|PubMed:9864343};
CC Vmax=7.5 pmol/min/mg enzyme with daunorubicin as substrate (at pH 7.5
CC and 30 degrees Celsius) {ECO:0000269|PubMed:9864343};
CC Vmax=19 pmol/min/mg enzyme with 13-dihydrocarminomycin as substrate
CC (at pH 7.5 and 30 degrees Celsius) {ECO:0000269|PubMed:9864343};
CC Vmax=1600 pmol/min/mg enzyme with 13-deoxydaunorubicin as substrate
CC (at pH 7.5 and 30 degrees Celsius) {ECO:0000269|PubMed:9864343};
CC Vmax=3900 pmol/min/mg enzyme with 13-dihydrodaunorubicin as substrate
CC (at pH 7.5 and 30 degrees Celsius) {ECO:0000269|PubMed:9864343};
CC Note=kcat is 0.63 sec(-1) for oxidation on 13-dihydrodaunorubicin.
CC kcat is 0.024 sec(-1) for hydroxylation on 13-deoxydaunorubicin. kcat
CC is 0.00026 sec(-1) for oxidation on 13-dihydrocarminomycin. kcat is
CC 0.00012 sec(-1) for hydroxylation on daunorubicin.;
CC pH dependence:
CC Optimum pH is 7.5. This optimal pH, slightly higher than normal
CC physiological conditions, may be necessary to ensure that the proper
CC ionic nature of the zwitterionic anthracycline substrates is
CC maintained for optimal recognition. {ECO:0000269|PubMed:9864343};
CC Temperature dependence:
CC Optimum temperature is 30 degrees Celsius.
CC {ECO:0000269|PubMed:9864343};
CC -!- PATHWAY: Antibiotic biosynthesis; daunorubicin biosynthesis.
CC -!- PATHWAY: Antibiotic biosynthesis; carminomycin biosynthesis.
CC -!- SUBUNIT: Monomer. {ECO:0000269|PubMed:9864343}.
CC -!- SUBCELLULAR LOCATION: Cytoplasm {ECO:0000250}.
CC -!- MISCELLANEOUS: Streptomyces sp. C5 produces significant quantities of
CC doxorubicin only when doxA is strongly overexpressed, but under normal
CC conditions, 14-hydroxylation of daunorubicin cannot favorably compete
CC with baumycin biosynthesis from daunorubicin. S.peucetius subsp.
CC caesius ATCC 27952, a mutant strain derived from S.peucetius ATCC
CC 29050, is the only organism reported to produce doxorubicin in vivo
CC (PubMed:9864343). {ECO:0000305|PubMed:9864343}.
CC -!- SIMILARITY: Belongs to the cytochrome P450 family. {ECO:0000305}.
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DR EMBL; U50973; AAB08049.1; -; Genomic_DNA.
DR AlphaFoldDB; Q59971; -.
DR SMR; Q59971; -.
DR KEGG; ag:AAB08049; -.
DR BioCyc; MetaCyc:MON-18172; -.
DR BRENDA; 1.14.13.181; 1284.
DR UniPathway; UPA00054; -.
DR UniPathway; UPA01040; -.
DR GO; GO:0005737; C:cytoplasm; IEA:UniProtKB-SubCell.
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; IDA:UniProtKB.
DR GO; GO:1901771; P:daunorubicin biosynthetic process; IDA:UniProtKB.
DR Gene3D; 1.10.630.10; -; 1.
DR InterPro; IPR001128; Cyt_P450.
DR InterPro; IPR002397; Cyt_P450_B.
DR InterPro; IPR017972; Cyt_P450_CS.
DR InterPro; IPR036396; Cyt_P450_sf.
DR Pfam; PF00067; p450; 1.
DR PRINTS; PR00359; BP450.
DR PRINTS; PR00385; P450.
DR SUPFAM; SSF48264; SSF48264; 1.
DR PROSITE; PS00086; CYTOCHROME_P450; 1.
PE 1: Evidence at protein level;
KW Antibiotic biosynthesis; Cytoplasm; Heme; Iron; Metal-binding;
KW Monooxygenase; NADP; Oxidoreductase.
FT CHAIN 1..422
FT /note="Cytochrome P-450 monooxygenase DoxA"
FT /id="PRO_0000425683"
FT BINDING 369
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"
SQ SEQUENCE 422 AA; 46096 MW; F4EADECA1D159052 CRC64;
MSGEAPRVAV DPFSCPMMTM QRKPEVHDAF REAGPVVEVN APAGGPAWVI TDDALAREVL
ADPRFVKDPD LAPTAWRGVD DGLDIPVPEL RPFTLIAVDG EDHRRLRRIH APAFNPRRLA
ERTDRIAAIA DRLLTELADS SDRSGEPAEL IGGFAYHFPL LVICELLGVP VTDPAMAREA
VGVLKALGLG GPQSAGGDGT DPAGDVPDTS ALESLLLEAV HAARRKDTRT MTRVLYERAQ
AEFGSVSDDQ LVYMITGLIF AGHDTTGSFL GFLLAEVLAG RLAADADGDA ISRFVEEALR
HHPPVPYTLW RFAATEVVIR GVRLPRGAPV LVDIEGTNTD GRHHDAPHAF HPDRPSRRRL
TFGDGPHYCI GEQLAQLESR TMIGVLRSRF PQARLAVPYE ELRWCRKGAQ TARLTDLPVW
LR
