AZOR1_PSEAE
ID AZOR1_PSEAE Reviewed; 212 AA.
AC Q9I5F3;
DT 26-JUL-2002, integrated into UniProtKB/Swiss-Prot.
DT 01-MAR-2001, sequence version 1.
DT 03-AUG-2022, entry version 110.
DE RecName: Full=FMN-dependent NAD(P)H:quinone oxidoreductase 1 {ECO:0000305};
DE EC=1.6.5.- {ECO:0000255|HAMAP-Rule:MF_01216, ECO:0000269|PubMed:24915188};
DE AltName: Full=Azo-dye reductase 1 {ECO:0000255|HAMAP-Rule:MF_01216};
DE AltName: Full=FMN-dependent NADH-azo compound oxidoreductase 1 {ECO:0000255|HAMAP-Rule:MF_01216};
DE AltName: Full=FMN-dependent NADH-azoreductase 1 {ECO:0000255|HAMAP-Rule:MF_01216};
DE EC=1.7.1.17 {ECO:0000255|HAMAP-Rule:MF_01216};
GN Name=azoR1 {ECO:0000255|HAMAP-Rule:MF_01216, ECO:0000303|PubMed:17904577};
GN OrderedLocusNames=PA0785;
OS Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM
OS 14847 / LMG 12228 / 1C / PRS 101 / PAO1).
OC Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales;
OC Pseudomonadaceae; Pseudomonas.
OX NCBI_TaxID=208964;
RN [1]
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RC STRAIN=ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C /
RC PRS 101 / PAO1;
RX PubMed=10984043; DOI=10.1038/35023079;
RA Stover C.K., Pham X.-Q.T., Erwin A.L., Mizoguchi S.D., Warrener P.,
RA Hickey M.J., Brinkman F.S.L., Hufnagle W.O., Kowalik D.J., Lagrou M.,
RA Garber R.L., Goltry L., Tolentino E., Westbrock-Wadman S., Yuan Y.,
RA Brody L.L., Coulter S.N., Folger K.R., Kas A., Larbig K., Lim R.M.,
RA Smith K.A., Spencer D.H., Wong G.K.-S., Wu Z., Paulsen I.T., Reizer J.,
RA Saier M.H. Jr., Hancock R.E.W., Lory S., Olson M.V.;
RT "Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic
RT pathogen.";
RL Nature 406:959-964(2000).
RN [2] {ECO:0007744|PDB:2V9C}
RP X-RAY CRYSTALLOGRAPHY (2.18 ANGSTROMS) IN COMPLEX WITH FMN AND METHYL RED,
RP FUNCTION, CATALYTIC ACTIVITY, COFACTOR, ACTIVITY REGULATION,
RP BIOPHYSICOCHEMICAL PROPERTIES, AND SUBUNIT.
RX PubMed=17904577; DOI=10.1016/j.jmb.2007.08.048;
RA Wang C.J., Hagemeier C., Rahman N., Lowe E., Noble M., Coughtrie M.,
RA Sim E., Westwood I.;
RT "Molecular cloning, characterisation and ligand-bound structure of an
RT azoreductase from Pseudomonas aeruginosa.";
RL J. Mol. Biol. 373:1213-1228(2007).
RN [3] {ECO:0007744|PDB:3KEG}
RP X-RAY CRYSTALLOGRAPHY (2.10 ANGSTROMS) OF MUTANT PHE-131 IN COMPLEX WITH
RP FMN AND METHYL RED, FUNCTION, CATALYTIC ACTIVITY, COFACTOR,
RP BIOPHYSICOCHEMICAL PROPERTIES, AND MUTAGENESIS OF TYR-131.
RX PubMed=20057057; DOI=10.1107/s1744309109044741;
RA Wang C.J., Laurieri N., Abuhammad A., Lowe E., Westwood I., Ryan A.,
RA Sim E.;
RT "Role of tyrosine 131 in the active site of paAzoR1, an azoreductase with
RT specificity for the inflammatory bowel disease prodrug balsalazide.";
RL Acta Crystallogr. F 66:2-7(2010).
RN [4] {ECO:0007744|PDB:3LT5}
RP X-RAY CRYSTALLOGRAPHY (2.30 ANGSTROMS) IN COMPLEX WITH FMN AND BALSALAZIDE.
RC STRAIN=ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C /
RC PRS 101 / PAO1;
RX PubMed=20417637; DOI=10.1016/j.jmb.2010.04.023;
RA Ryan A., Laurieri N., Westwood I., Wang C.J., Lowe E., Sim E.;
RT "A novel mechanism for azoreduction.";
RL J. Mol. Biol. 400:24-37(2010).
RN [5] {ECO:0007744|PDB:3R6W}
RP X-RAY CRYSTALLOGRAPHY (2.08 ANGSTROMS) IN COMPLEX WITH FMN AND
RP NITROFURAZONE, FUNCTION, AND BIOPHYSICOCHEMICAL PROPERTIES.
RX PubMed=22355582; DOI=10.1038/srep00063;
RA Ryan A., Kaplan E., Laurieri N., Lowe E., Sim E.;
RT "Activation of nitrofurazone by azoreductases: multiple activities in one
RT enzyme.";
RL Sci. Rep. 1:63-63(2011).
RN [6] {ECO:0007744|PDB:4N65, ECO:0007744|PDB:4N9Q}
RP X-RAY CRYSTALLOGRAPHY (1.82 ANGSTROMS) IN COMPLEXES WITH FMN;
RP ANTHRAQUINONE-2-SULPHONATE AND UBIQUINONE-1, FUNCTION, AND CATALYTIC
RP ACTIVITY.
RX PubMed=24915188; DOI=10.1371/journal.pone.0098551;
RA Ryan A., Kaplan E., Nebel J.C., Polycarpou E., Crescente V., Lowe E.,
RA Preston G.M., Sim E.;
RT "Identification of NAD(P)H quinone oxidoreductase activity in azoreductases
RT from P. aeruginosa: azoreductases and NAD(P)H quinone oxidoreductases
RT belong to the same FMN-dependent superfamily of enzymes.";
RL PLoS ONE 9:e98551-e98551(2014).
CC -!- FUNCTION: Quinone reductase that provides resistance to thiol-specific
CC stress caused by electrophilic quinones (PubMed:24915188). Shows a
CC preference for benzoquinones (PubMed:24915188).
CC {ECO:0000269|PubMed:24915188}.
CC -!- FUNCTION: Also exhibits azoreductase activity. Catalyzes the reductive
CC cleavage of the azo bond in aromatic azo compounds to the corresponding
CC amines (PubMed:17904577, PubMed:20057057). NADPH is the preferred
CC electron donor for azoreductase activity, but it can also use NADH
CC (PubMed:17904577). Can reduce different classes of azo dyes, including
CC the common azo dyes methyl red and p-aminoazobenzene sulfonamide
CC (PAABSA) (PubMed:17904577, PubMed:20057057). Can activate several azo
CC pro-drugs used in the treatment of inflammatory bowel disease (IBD),
CC including balsalazide, sulfasalazine and olsalazine (PubMed:17904577,
CC PubMed:20057057). Also acts as a nitrodeductase, and can reduce and
CC hence activate the nitroaromatic drug nitrofurazone, a broad spectrum
CC antibiotic (PubMed:22355582). {ECO:0000269|PubMed:17904577,
CC ECO:0000269|PubMed:20057057, ECO:0000269|PubMed:22355582}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=2 a quinone + H(+) + NADPH = 2 a 1,4-benzosemiquinone +
CC NADP(+); Xref=Rhea:RHEA:14269, ChEBI:CHEBI:15378, ChEBI:CHEBI:57783,
CC ChEBI:CHEBI:58349, ChEBI:CHEBI:132124, ChEBI:CHEBI:134225;
CC Evidence={ECO:0000269|PubMed:24915188};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=2 a quinone + H(+) + NADH = 2 a 1,4-benzosemiquinone + NAD(+);
CC Xref=Rhea:RHEA:65952, ChEBI:CHEBI:15378, ChEBI:CHEBI:57540,
CC ChEBI:CHEBI:57945, ChEBI:CHEBI:132124, ChEBI:CHEBI:134225;
CC Evidence={ECO:0000255|HAMAP-Rule:MF_01216};
CC -!- CATALYTIC ACTIVITY:
CC Reaction=anthranilate + N,N-dimethyl-1,4-phenylenediamine + 2 NAD(+) =
CC 2-(4-dimethylaminophenyl)diazenylbenzoate + 2 H(+) + 2 NADH;
CC Xref=Rhea:RHEA:55872, ChEBI:CHEBI:15378, ChEBI:CHEBI:15783,
CC ChEBI:CHEBI:16567, ChEBI:CHEBI:57540, ChEBI:CHEBI:57945,
CC ChEBI:CHEBI:71579; EC=1.7.1.17; Evidence={ECO:0000255|HAMAP-
CC Rule:MF_01216, ECO:0000269|PubMed:17904577,
CC ECO:0000269|PubMed:20057057};
CC PhysiologicalDirection=right-to-left; Xref=Rhea:RHEA:55874;
CC Evidence={ECO:0000269|PubMed:17904577, ECO:0000269|PubMed:20057057};
CC -!- COFACTOR:
CC Name=FMN; Xref=ChEBI:CHEBI:58210;
CC Evidence={ECO:0000255|HAMAP-Rule:MF_01216,
CC ECO:0000269|PubMed:17904577, ECO:0000269|PubMed:20057057};
CC Note=Binds 1 FMN per subunit. {ECO:0000255|HAMAP-Rule:MF_01216,
CC ECO:0000269|PubMed:17904577, ECO:0000269|PubMed:20057057};
CC -!- ACTIVITY REGULATION: Azoreductase activity increases with salt
CC strength. {ECO:0000269|PubMed:17904577}.
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=76 uM for methyl red (in the presence of NADPH)
CC {ECO:0000269|PubMed:17904577};
CC KM=92.7 uM for methyl red (in the presence of NADPH)
CC {ECO:0000269|PubMed:20057057};
CC KM=69 uM for sulfasalazine (in the presence of NADPH)
CC {ECO:0000269|PubMed:17904577};
CC KM=124 uM for balsalazide (in the presence of NADPH)
CC {ECO:0000269|PubMed:17904577};
CC KM=98.6 uM for balsalazide (in the presence of NADPH)
CC {ECO:0000269|PubMed:20057057};
CC KM=104 uM for olsalazine (in the presence of NADPH)
CC {ECO:0000269|PubMed:17904577};
CC KM=15.7 uM for nitrofurazone {ECO:0000269|PubMed:22355582};
CC KM=464 uM for NADH (in the presence of methyl red)
CC {ECO:0000269|PubMed:17904577};
CC KM=538 uM for NADH (in the presence of methyl red)
CC {ECO:0000269|PubMed:20057057};
CC KM=1100 uM for NADPH (in the presence of methyl red)
CC {ECO:0000269|PubMed:17904577};
CC KM=1197 uM for NADPH (in the presence of methyl red)
CC {ECO:0000269|PubMed:20057057};
CC Vmax=28 umol/sec/mg enzyme with methyl red as substrate (in the
CC presence of NADPH) {ECO:0000269|PubMed:17904577};
CC Vmax=28 umol/sec/mg enzyme with sulfasalazine as substrate (in the
CC presence of NADPH) {ECO:0000269|PubMed:17904577};
CC Vmax=81 umol/sec/mg enzyme with balsalazide as substrate (in the
CC presence of NADPH) {ECO:0000269|PubMed:17904577};
CC Vmax=5.4 umol/sec/mg enzyme with olsalazine as substrate (in the
CC presence of NADPH) {ECO:0000269|PubMed:17904577};
CC Vmax=22 umol/sec/mg enzyme with NADH as substrate (in the presence of
CC methyl red) {ECO:0000269|PubMed:17904577};
CC Vmax=74 umol/sec/mg enzyme with NADPH as substrate (in the presence
CC of methyl red) {ECO:0000269|PubMed:17904577};
CC Note=kcat is 13 sec(-1) with methyl red as substrate. kcat is 13
CC sec(-1) with sulfasalazine as substrate. kcat is 37 sec(-1) with
CC balsalazide as substrate. kcat is 2.5 sec(-1) with olsalazine as
CC substrate. kcat is 10 sec(-1) with NADH as substrate. kcat is 34
CC sec(-1) with NADPH as substrate. {ECO:0000269|PubMed:17904577};
CC -!- SUBUNIT: Homodimer (PubMed:17904577). Homotetramer formed by a dimer of
CC dimers when the ionic strength is high (PubMed:17904577).
CC {ECO:0000269|PubMed:17904577}.
CC -!- MISCELLANEOUS: Rate of quinone reduction is higher than reduction of
CC azo substrates, suggesting the enzyme is better suited for carrying out
CC quinone rather than azo reduction. {ECO:0000269|PubMed:24915188}.
CC -!- SIMILARITY: Belongs to the azoreductase type 1 family.
CC {ECO:0000255|HAMAP-Rule:MF_01216}.
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DR EMBL; AE004091; AAG04174.1; -; Genomic_DNA.
DR PIR; H83547; H83547.
DR RefSeq; NP_249476.1; NC_002516.2.
DR RefSeq; WP_003114189.1; NZ_QZGE01000007.1.
DR PDB; 2V9C; X-ray; 2.18 A; A/B=1-212.
DR PDB; 3KEG; X-ray; 2.10 A; A/B=1-212.
DR PDB; 3LT5; X-ray; 2.30 A; A/B=1-212.
DR PDB; 3R6W; X-ray; 2.08 A; A/B=1-212.
DR PDB; 4N65; X-ray; 1.82 A; A/B=1-212.
DR PDB; 4N9Q; X-ray; 2.00 A; A/B=1-212.
DR PDBsum; 2V9C; -.
DR PDBsum; 3KEG; -.
DR PDBsum; 3LT5; -.
DR PDBsum; 3R6W; -.
DR PDBsum; 4N65; -.
DR PDBsum; 4N9Q; -.
DR AlphaFoldDB; Q9I5F3; -.
DR SMR; Q9I5F3; -.
DR STRING; 287.DR97_1198; -.
DR DrugBank; DB08209; Methyl red.
DR PaxDb; Q9I5F3; -.
DR DNASU; 882036; -.
DR EnsemblBacteria; AAG04174; AAG04174; PA0785.
DR GeneID; 882036; -.
DR KEGG; pae:PA0785; -.
DR PATRIC; fig|208964.12.peg.816; -.
DR PseudoCAP; PA0785; -.
DR HOGENOM; CLU_088964_0_0_6; -.
DR InParanoid; Q9I5F3; -.
DR OMA; WIHAAFT; -.
DR PhylomeDB; Q9I5F3; -.
DR BioCyc; PAER208964:G1FZ6-798-MON; -.
DR BRENDA; 1.7.1.6; 5087.
DR EvolutionaryTrace; Q9I5F3; -.
DR Proteomes; UP000002438; Chromosome.
DR GO; GO:0009055; F:electron transfer activity; IEA:UniProtKB-UniRule.
DR GO; GO:0010181; F:FMN binding; IEA:UniProtKB-UniRule.
DR GO; GO:0003960; F:NADPH:quinone reductase activity; IEA:RHEA.
DR GO; GO:0016652; F:oxidoreductase activity, acting on NAD(P)H, NAD(P) as acceptor; IEA:UniProtKB-UniRule.
DR GO; GO:0016655; F:oxidoreductase activity, acting on NAD(P)H, quinone or similar compound as acceptor; IDA:PseudoCAP.
DR Gene3D; 3.40.50.360; -; 1.
DR HAMAP; MF_01216; Azoreductase_type1; 1.
DR InterPro; IPR003680; Flavodoxin_fold.
DR InterPro; IPR029039; Flavoprotein-like_sf.
DR InterPro; IPR023048; NADH:quinone_OxRdtase_FMN_depd.
DR Pfam; PF02525; Flavodoxin_2; 1.
DR SUPFAM; SSF52218; SSF52218; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Flavoprotein; FMN; NAD; Oxidoreductase; Reference proteome.
FT CHAIN 1..212
FT /note="FMN-dependent NAD(P)H:quinone oxidoreductase 1"
FT /id="PRO_0000166347"
FT BINDING 10
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01216,
FT ECO:0000269|PubMed:17904577, ECO:0000269|PubMed:20057057,
FT ECO:0000269|PubMed:20417637, ECO:0000269|PubMed:22355582,
FT ECO:0000269|PubMed:24915188, ECO:0007744|PDB:2V9C,
FT ECO:0007744|PDB:3KEG, ECO:0007744|PDB:3LT5,
FT ECO:0007744|PDB:3R6W, ECO:0007744|PDB:4N65,
FT ECO:0007744|PDB:4N9Q"
FT BINDING 16..18
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01216,
FT ECO:0000269|PubMed:17904577, ECO:0000269|PubMed:20057057,
FT ECO:0000269|PubMed:20417637, ECO:0000269|PubMed:22355582,
FT ECO:0000269|PubMed:24915188, ECO:0007744|PDB:2V9C,
FT ECO:0007744|PDB:3KEG, ECO:0007744|PDB:3LT5,
FT ECO:0007744|PDB:3R6W, ECO:0007744|PDB:4N65,
FT ECO:0007744|PDB:4N9Q"
FT BINDING 97..100
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01216,
FT ECO:0000269|PubMed:17904577, ECO:0000269|PubMed:20057057,
FT ECO:0000269|PubMed:20417637, ECO:0000269|PubMed:22355582,
FT ECO:0000269|PubMed:24915188, ECO:0007744|PDB:2V9C,
FT ECO:0007744|PDB:3KEG, ECO:0007744|PDB:3LT5,
FT ECO:0007744|PDB:3R6W, ECO:0007744|PDB:4N65,
FT ECO:0007744|PDB:4N9Q"
FT BINDING 99
FT /ligand="substrate"
FT /evidence="ECO:0000305|PubMed:20417637,
FT ECO:0000305|PubMed:22355582, ECO:0007744|PDB:3LT5,
FT ECO:0007744|PDB:3R6W"
FT BINDING 131
FT /ligand="substrate"
FT /evidence="ECO:0000305|PubMed:20417637,
FT ECO:0000305|PubMed:22355582, ECO:0007744|PDB:3LT5,
FT ECO:0007744|PDB:3R6W"
FT BINDING 145..148
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000255|HAMAP-Rule:MF_01216,
FT ECO:0000269|PubMed:17904577, ECO:0000269|PubMed:20057057,
FT ECO:0000269|PubMed:20417637, ECO:0000269|PubMed:22355582,
FT ECO:0000269|PubMed:24915188, ECO:0007744|PDB:2V9C,
FT ECO:0007744|PDB:3KEG, ECO:0007744|PDB:3LT5,
FT ECO:0007744|PDB:3R6W, ECO:0007744|PDB:4N65,
FT ECO:0007744|PDB:4N9Q"
FT BINDING 187
FT /ligand="FMN"
FT /ligand_id="ChEBI:CHEBI:58210"
FT /evidence="ECO:0000269|PubMed:20417637,
FT ECO:0000269|PubMed:22355582, ECO:0000269|PubMed:24915188,
FT ECO:0007744|PDB:3LT5, ECO:0007744|PDB:3R6W,
FT ECO:0007744|PDB:4N65, ECO:0007744|PDB:4N9Q"
FT BINDING 188
FT /ligand="substrate"
FT /evidence="ECO:0000305|PubMed:20057057,
FT ECO:0000305|PubMed:20417637, ECO:0007744|PDB:3KEG,
FT ECO:0007744|PDB:3LT5"
FT SITE 131
FT /note="Important in the architecture of the active site"
FT /evidence="ECO:0000269|PubMed:20057057"
FT MUTAGEN 131
FT /note="Y->F: 2-fold increase in specific activity towards
FT methyl red and 20% decrease in specific activity towards
FT balsalazide. 2.5-fold increase in the kcat with NADH as
FT substrate."
FT /evidence="ECO:0000269|PubMed:20057057"
FT STRAND 3..8
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 17..32
FT /evidence="ECO:0007829|PDB:4N65"
FT STRAND 37..42
FT /evidence="ECO:0007829|PDB:4N65"
FT STRAND 44..46
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 53..59
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 64..66
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 69..86
FT /evidence="ECO:0007829|PDB:4N65"
FT STRAND 89..96
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 104..113
FT /evidence="ECO:0007829|PDB:4N65"
FT TURN 116..118
FT /evidence="ECO:0007829|PDB:4N65"
FT STRAND 119..123
FT /evidence="ECO:0007829|PDB:4N65"
FT STRAND 130..133
FT /evidence="ECO:0007829|PDB:4N65"
FT STRAND 139..149
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 158..160
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 164..173
FT /evidence="ECO:0007829|PDB:4N65"
FT STRAND 179..185
FT /evidence="ECO:0007829|PDB:4N65"
FT TURN 187..189
FT /evidence="ECO:0007829|PDB:4N65"
FT HELIX 194..211
FT /evidence="ECO:0007829|PDB:4N65"
SQ SEQUENCE 212 AA; 23050 MW; 356AF121972823B2 CRC64;
MSRILAVHAS PRGERSQSRR LAEVFLAAYR EAHPQARVAR REVGRVPLPA VTEAFVAAAF
HPQPEQRSLA MQADLALSDQ LVGELFDSDL LVISTPMYNF SVPSGLKAWI DQIVRLGVTF
DFVLDNGVAQ YRPLLRGKRA LIVTSRGGHG FGPGGENQAM NHADPWLRTA LGFIGIDEVT
VVAAEGEESG GRSFEDSCDE AEQRLLALAR SA
