PHHY_PSEFL
ID PHHY_PSEFL Reviewed; 394 AA.
AC P00438;
DT 21-JUL-1986, integrated into UniProtKB/Swiss-Prot.
DT 01-APR-1993, sequence version 2.
DT 03-AUG-2022, entry version 128.
DE RecName: Full=p-hydroxybenzoate hydroxylase {ECO:0000303|PubMed:1459126};
DE Short=PHBH {ECO:0000303|PubMed:1459126};
DE Short=PHBHase {ECO:0000303|PubMed:3351945};
DE EC=1.14.13.2 {ECO:0000269|PubMed:10025942, ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:1459126, ECO:0000269|PubMed:7628466, ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477, ECO:0000269|PubMed:9694855};
DE AltName: Full=4-hydroxybenzoate 3-monooxygenase {ECO:0000305};
GN Name=pobA;
OS Pseudomonas fluorescens.
OC Bacteria; Proteobacteria; Gammaproteobacteria; Pseudomonadales;
OC Pseudomonadaceae; Pseudomonas.
OX NCBI_TaxID=294;
RN [1]
RP NUCLEOTIDE SEQUENCE [GENOMIC DNA], FUNCTION, CATALYTIC ACTIVITY,
RP MUTAGENESIS OF CYS-116 AND ARG-214, BIOPHYSICOCHEMICAL PROPERTIES, AND
RP COFACTOR.
RX PubMed=1459126; DOI=10.1111/j.1432-1033.1992.tb17436.x;
RA van Berkel W., Westphal A., Eschrich K., Eppink M., de Kok A.;
RT "Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate
RT hydroxylase from Pseudomonas fluorescens.";
RL Eur. J. Biochem. 210:411-419(1992).
RN [2]
RP PROTEIN SEQUENCE.
RX PubMed=6809053; DOI=10.1016/0167-4838(82)90170-4;
RA Weijer W.J., Hofsteenge J., Vereijken J.M., Jekel P.A., Beintema J.J.;
RT "Primary structure of p-hydroxybenzoate hydroxylase from Pseudomonas
RT fluorescens.";
RL Biochim. Biophys. Acta 704:385-388(1982).
RN [3]
RP PROTEIN SEQUENCE OF 111-138 AND 270-280.
RX PubMed=6780352;
RA Hofsteenge J., Vereijken J.M., Weijer W.J., Beintema J.J., Wierenga R.K.,
RA Drenth J.;
RT "Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase
RT from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides;
RT interactions of the protein with flavin adenine dinucleotide.";
RL Eur. J. Biochem. 113:141-150(1980).
RN [4]
RP PROTEIN SEQUENCE OF 1-52; 53-65 AND 66-110.
RX PubMed=6780353; DOI=10.1111/j.1432-1033.1980.tb06149.x;
RA Vereijken J.M., Hofsteenge J., Bak H.J., Beintema J.J.;
RT "The amino-acid sequence of the three smallest CNBr peptides from p-
RT hydroxybenzoate hydroxylase from Pseudomonas fluorescens.";
RL Eur. J. Biochem. 113:151-157(1980).
RN [5]
RP PROTEIN SEQUENCE OF CNBR PEPTIDES AND TERTIARY STRUCTURE.
RX PubMed=6406229; DOI=10.1111/j.1432-1033.1983.tb07433.x;
RA Hofsteenge J., Weijer W.J., Jekel P.A., Beintema J.J.;
RT "p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 1. Completion
RT of the elucidation of the primary structure.";
RL Eur. J. Biochem. 133:91-108(1983).
RN [6]
RP PROTEIN SEQUENCE OF CNBR PEPTIDES, AND FAD BINDING SITE.
RX PubMed=6406227; DOI=10.1111/j.1432-1033.1983.tb07435.x;
RA Weijer W.J., Hofsteenge J., Beintema J.J., Wierenga R.K., Drenth J.;
RT "p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. 2. Fitting of
RT the amino-acid sequence to the tertiary structure.";
RL Eur. J. Biochem. 133:109-118(1983).
RN [7]
RP X-RAY CRYSTALLOGRAPHY (2.5 ANGSTROMS) IN COMPLEX WITH SUBSTRATE AND FAD,
RP AND SUBUNIT.
RX PubMed=40036; DOI=10.1016/0022-2836(79)90301-2;
RA Wierenga R.K., de Jong R.J., Kalk K.H., Hol W.G.J., Drenth J.;
RT "Crystal structure of p-hydroxybenzoate hydroxylase.";
RL J. Mol. Biol. 131:55-73(1979).
RN [8]
RP X-RAY CRYSTALLOGRAPHY (2.3 ANGSTROMS) OF REDUCED FORM IN COMPLEX WITH
RP SUBSTRATE AND FAD, AND COFACTOR.
RX PubMed=1409567; DOI=10.1002/prot.340140205;
RA Schreuder H.A., van der Laan J.M., Swarte M.B.A., Kalk K.H., Hol W.G.J.,
RA Drenth J.;
RT "Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase
RT refined at 2.3-A resolution.";
RL Proteins 14:178-190(1992).
RN [9]
RP X-RAY CRYSTALLOGRAPHY (2.30 ANGSTROMS) IN COMPLEX WITH SUBSTRATE ANALOG AND
RP FAD, FUNCTION, COFACTOR, AND SUBUNIT.
RX PubMed=3351945; DOI=10.1016/0022-2836(88)90307-5;
RA Schreuder H.A., van der Laan J.M., Hol W.G., Drenth J.;
RT "Crystal structure of p-hydroxybenzoate hydroxylase complexed with its
RT reaction product 3,4-dihydroxybenzoate.";
RL J. Mol. Biol. 199:637-648(1988).
RN [10]
RP X-RAY CRYSTALLOGRAPHY (2.70 ANGSTROMS) IN COMPLEX WITH SUBSTRATE AND FAD
RP ANALOG, FUNCTION, COFACTOR, AND SUBUNIT.
RX PubMed=2819062; DOI=10.1021/bi00444a011;
RA van der Laan J.M., Schreuder H.A., Swarte M.B., Wierenga R.K., Kalk K.H.,
RA Hol W.G., Drenth J.;
RT "The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the
RT active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic
RT investigation.";
RL Biochemistry 28:7199-7205(1989).
RN [11]
RP X-RAY CRYSTALLOGRAPHY (1.90 ANGSTROMS) IN COMPLEX WITH SUBSTRATE AND FAD
RP ANALOG, AND SUBUNIT.
RX PubMed=2553983; DOI=10.1016/0022-2836(89)90158-7;
RA Schreuder H.A., Prick P.A., Wierenga R.K., Vriend G., Wilson K.S.,
RA Hol W.G., Drenth J.;
RT "Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex
RT refined at 1.9 A resolution. Analysis of the enzyme-substrate and enzyme-
RT product complexes.";
RL J. Mol. Biol. 208:679-696(1989).
RN [12]
RP X-RAY CRYSTALLOGRAPHY (2.30 ANGSTROMS) OF WILD-TYPE AND MUTANT ALA-222 IN
RP COMPLEX WITH SUBSTRATE ANALOGS AND FAD, FUNCTION, MUTAGENESIS OF TYR-222,
RP AND SUBUNIT.
RX PubMed=7520279; DOI=10.1021/bi00199a044;
RA Schreuder H.A., Mattevi A., Obmolova G., Kalk K.H., Hol W.G.,
RA van der Bolt F.J., van Berkel W.J.;
RT "Crystal structures of wild-type p-hydroxybenzoate hydroxylase complexed
RT with 4-aminobenzoate,2,4-dihydroxybenzoate, and 2-hydroxy-4-aminobenzoate
RT and of the Tyr222Ala mutant complexed with 2-hydroxy-4-aminobenzoate.
RT Evidence for a proton channel and a new binding mode of the flavin ring.";
RL Biochemistry 33:10161-10170(1994).
RN [13]
RP X-RAY CRYSTALLOGRAPHY (2.10 ANGSTROMS) IN COMPLEX WITH SUBSTRATE AND FAD
RP ANALOG, FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL PROPERTIES, AND
RP SUBUNIT.
RX PubMed=7756982; DOI=10.1002/pro.5560031210;
RA van Berkel W.J., Eppink M.H., Schreuder H.A.;
RT "Crystal structure of p-hydroxybenzoate hydroxylase reconstituted with the
RT modified FAD present in alcohol oxidase from methylotrophic yeasts:
RT evidence for an arabinoflavin.";
RL Protein Sci. 3:2245-2253(1994).
RN [14]
RP X-RAY CRYSTALLOGRAPHY (2.20 ANGSTROMS) OF MUTANT LYS-44 IN COMPLEX WITH
RP SUBSTRATE AND FAD, FUNCTION, CATALYTIC ACTIVITY, MUTAGENESIS OF ARG-44,
RP COFACTOR, AND SUBUNIT.
RX PubMed=7628466; DOI=10.1111/j.1432-1033.1995.0157f.x;
RA Eppink M.H., Schreuder H.A., Van Berkel W.J.;
RT "Structure and function of mutant Arg44Lys of 4-hydroxybenzoate hydroxylase
RT implications for NADPH binding.";
RL Eur. J. Biochem. 231:157-165(1995).
RN [15]
RP X-RAY CRYSTALLOGRAPHY (2.20 ANGSTROMS) OF MUTANT LYS-42 AND SER-42 IN
RP COMPLEX WITH SUBSTRATE AND FAD, FUNCTION, CATALYTIC ACTIVITY,
RP BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF ARG-42, COFACTOR, AND
RP SUBUNIT.
RX PubMed=9578477; DOI=10.1046/j.1432-1327.1998.2530194.x;
RA Eppink M.H., Schreuder H.A., van Berkel W.J.;
RT "Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas
RT fluorescens reveal that Arg42 is essential for NADPH binding.";
RL Eur. J. Biochem. 253:194-201(1998).
RN [16]
RP X-RAY CRYSTALLOGRAPHY (2.00 ANGSTROMS) OF MUTANTS ARG-162 AND THR-269 IN
RP COMPLEX WITH SUBSTRATE AND FAD, FUNCTION, CATALYTIC ACTIVITY,
RP BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF HIS-162 AND ARG-269,
RP COFACTOR, AND SUBUNIT.
RX PubMed=9694855; DOI=10.1074/jbc.273.33.21031;
RA Eppink M.H., Schreuder H.A., van Berkel W.J.;
RT "Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested
RT by kinetic, crystallographic and modeling studies of histidine 162 and
RT arginine 269 variants.";
RL J. Biol. Chem. 273:21031-21039(1998).
RN [17]
RP X-RAY CRYSTALLOGRAPHY (2.00 ANGSTROMS) OF ALA-161 AND SER-166 IN COMPLEX
RP WITH SUBSTRATE AND FAD, FUNCTION, CATALYTIC ACTIVITY, BIOPHYSICOCHEMICAL
RP PROPERTIES, MUTAGENESIS OF PHE-161 AND ARG-166, COFACTOR, AND SUBUNIT.
RX PubMed=10025942; DOI=10.1016/s0014-5793(98)01726-8;
RA Eppink M.H., Bunthol C., Schreuder H.A., van Berkel W.J.;
RT "Phe161 and Arg166 variants of p-hydroxybenzoate hydroxylase. Implications
RT for NADPH recognition and structural stability.";
RL FEBS Lett. 443:251-255(1999).
RN [18]
RP X-RAY CRYSTALLOGRAPHY (2.40 ANGSTROMS) OF 1-392 OF MUTANTS ARG-34; THR-34
RP AND GLU-38 IN COMPLEX WITH SUBSTRATE AND FAD, FUNCTION, CATALYTIC ACTIVITY,
RP BIOPHYSICOCHEMICAL PROPERTIES, MUTAGENESIS OF ARG-33; GLN-34 AND TYR-38,
RP COFACTOR, AND SUBUNIT.
RX PubMed=10493859; DOI=10.1006/jmbi.1999.3015;
RA Eppink M.H., Overkamp K.M., Schreuder H.A., Van Berkel W.J.;
RT "Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.";
RL J. Mol. Biol. 292:87-96(1999).
CC -!- FUNCTION: Catalyzes the incorporation of an atom of dioxygen into p-
CC hydroxybenzoate (p-OHB) to form 3,4-dihydroxybenzoate (3,4DOHB). The
CC reaction occurs in two parts: reduction of the flavin adenine
CC dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine
CC dinucleotide phosphate (NADPH) in response to binding p-hydroxybenzoate
CC to the enzyme and oxidation of reduced FAD with oxygen to form a
CC hydroperoxide, which then oxygenates p-hydroxybenzoate.
CC {ECO:0000269|PubMed:10025942, ECO:0000269|PubMed:10493859,
CC ECO:0000269|PubMed:1459126, ECO:0000269|PubMed:2819062,
CC ECO:0000269|PubMed:3351945, ECO:0000269|PubMed:7520279,
CC ECO:0000269|PubMed:7628466, ECO:0000269|PubMed:7756982,
CC ECO:0000269|PubMed:9578477, ECO:0000269|PubMed:9694855}.
CC -!- CATALYTIC ACTIVITY:
CC Reaction=4-hydroxybenzoate + H(+) + NADPH + O2 = 3,4-dihydroxybenzoate
CC + H2O + NADP(+); Xref=Rhea:RHEA:19477, ChEBI:CHEBI:15377,
CC ChEBI:CHEBI:15378, ChEBI:CHEBI:15379, ChEBI:CHEBI:17879,
CC ChEBI:CHEBI:36241, ChEBI:CHEBI:57783, ChEBI:CHEBI:58349;
CC EC=1.14.13.2; Evidence={ECO:0000269|PubMed:10025942,
CC ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:1459126,
CC ECO:0000269|PubMed:7628466, ECO:0000269|PubMed:7756982,
CC ECO:0000269|PubMed:9578477, ECO:0000269|PubMed:9694855};
CC -!- COFACTOR:
CC Name=FAD; Xref=ChEBI:CHEBI:57692;
CC Evidence={ECO:0000269|PubMed:10025942, ECO:0000269|PubMed:10493859,
CC ECO:0000269|PubMed:1409567, ECO:0000269|PubMed:1459126,
CC ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
CC ECO:0000269|PubMed:7628466, ECO:0000269|PubMed:9578477,
CC ECO:0000269|PubMed:9694855};
CC Note=Binds 1 FAD per subunit. {ECO:0000269|PubMed:10025942,
CC ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2819062,
CC ECO:0000269|PubMed:3351945, ECO:0000269|PubMed:7628466,
CC ECO:0000269|PubMed:9578477, ECO:0000269|PubMed:9694855};
CC -!- BIOPHYSICOCHEMICAL PROPERTIES:
CC Kinetic parameters:
CC KM=15 uM for p-OHB {ECO:0000269|PubMed:9578477};
CC KM=15 uM for p-OHB (at pH 6) {ECO:0000269|PubMed:10025942,
CC ECO:0000269|PubMed:10493859};
CC KM=20 uM for p-OHB {ECO:0000269|PubMed:7756982,
CC ECO:0000269|PubMed:9694855};
CC KM=25 uM for p-OHB (at 25 degrees Celsius)
CC {ECO:0000269|PubMed:1459126};
CC KM=30 uM for NADPH {ECO:0000269|PubMed:9694855};
CC KM=30 uM for p-OHB (at 6 degrees Celsius)
CC {ECO:0000269|PubMed:1459126};
CC KM=34 uM for NADPH (at pH 6) {ECO:0000269|PubMed:10025942,
CC ECO:0000269|PubMed:10493859};
CC KM=40 uM for NADPH (at 6 degrees Celsius)
CC {ECO:0000269|PubMed:1459126};
CC KM=50 uM for NADPH (at 25 degrees Celsius)
CC {ECO:0000269|PubMed:1459126};
CC KM=50 uM for NADPH {ECO:0000269|PubMed:9578477};
CC KM=70 uM for NADPH {ECO:0000269|PubMed:7756982};
CC Note=kcat is 55 sec(-1) for hydroxylase activity (PubMed:9578477,
CC PubMed:9694855). kcat is 55 sec(-1) for hydroxylase activity (at 25
CC degrees Celsius) (PubMed:1459126). kcat is 55 sec(-1) for hydroxylase
CC activity (at pH 8) (PubMed:10025942, PubMed:10493859). kcat is 9
CC sec(-1) for hydroxylase activity (at 6 degrees Celsius)
CC (PubMed:1459126). {ECO:0000269|PubMed:10025942,
CC ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:1459126,
CC ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
CC ECO:0000269|PubMed:9694855};
CC -!- PATHWAY: Aromatic compound metabolism; benzoate degradation via
CC hydroxylation; 3,4-dihydroxybenzoate from benzoate: step 2/2.
CC {ECO:0000305}.
CC -!- SUBUNIT: Homodimer. {ECO:0000269|PubMed:10025942,
CC ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
CC ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
CC ECO:0000269|PubMed:40036, ECO:0000269|PubMed:7520279,
CC ECO:0000269|PubMed:7628466, ECO:0000269|PubMed:7756982,
CC ECO:0000269|PubMed:9578477, ECO:0000269|PubMed:9694855}.
CC -!- SIMILARITY: Belongs to the aromatic-ring hydroxylase family.
CC {ECO:0000305}.
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DR EMBL; X68438; CAA48483.1; -; Genomic_DNA.
DR PIR; A90643; WHPSBF.
DR PDB; 1BF3; X-ray; 2.20 A; A=1-394.
DR PDB; 1BGJ; X-ray; 3.00 A; A=1-394.
DR PDB; 1BGN; X-ray; 2.00 A; A=1-394.
DR PDB; 1BKW; X-ray; 2.20 A; A=1-394.
DR PDB; 1CC4; X-ray; 2.00 A; A=1-394.
DR PDB; 1CC6; X-ray; 2.20 A; A=1-394.
DR PDB; 1CJ2; X-ray; 2.80 A; A=1-391.
DR PDB; 1CJ3; X-ray; 2.50 A; A=1-392.
DR PDB; 1CJ4; X-ray; 2.40 A; A=1-392.
DR PDB; 1PBB; X-ray; 2.50 A; A=1-394.
DR PDB; 1PBC; X-ray; 2.80 A; A=1-394.
DR PDB; 1PBD; X-ray; 2.30 A; A=1-394.
DR PDB; 1PBE; X-ray; 1.90 A; A=1-394.
DR PDB; 1PBF; X-ray; 2.70 A; A=1-394.
DR PDB; 1PDH; X-ray; 2.10 A; A=1-394.
DR PDB; 1PHH; X-ray; 2.30 A; A=1-394.
DR PDB; 2PHH; X-ray; 2.70 A; A=1-394.
DR PDBsum; 1BF3; -.
DR PDBsum; 1BGJ; -.
DR PDBsum; 1BGN; -.
DR PDBsum; 1BKW; -.
DR PDBsum; 1CC4; -.
DR PDBsum; 1CC6; -.
DR PDBsum; 1CJ2; -.
DR PDBsum; 1CJ3; -.
DR PDBsum; 1CJ4; -.
DR PDBsum; 1PBB; -.
DR PDBsum; 1PBC; -.
DR PDBsum; 1PBD; -.
DR PDBsum; 1PBE; -.
DR PDBsum; 1PBF; -.
DR PDBsum; 1PDH; -.
DR PDBsum; 1PHH; -.
DR PDBsum; 2PHH; -.
DR AlphaFoldDB; P00438; -.
DR SMR; P00438; -.
DR BindingDB; P00438; -.
DR ChEMBL; CHEMBL3675; -.
DR DrugBank; DB02839; 2,4-Dihydroxybenzoic Acid.
DR DrugBank; DB04242; 4-hydroxybenzoic acid.
DR DrugBank; DB02059; Adenosine-5-Diphosphoribose.
DR DrugBank; DB02362; Aminobenzoic acid.
DR DrugBank; DB03147; Flavin adenine dinucleotide.
DR BioCyc; MetaCyc:MON-11534; -.
DR BRENDA; 1.14.13.2; 5121.
DR UniPathway; UPA00156; UER00257.
DR EvolutionaryTrace; P00438; -.
DR GO; GO:0106356; F:4-hydroxybenzoate 3-monooxygenase [NADPH] activity; IEA:RHEA.
DR GO; GO:0018659; F:4-hydroxybenzoate 3-monooxygenase activity; IDA:UniProtKB.
DR GO; GO:0071949; F:FAD binding; IDA:UniProtKB.
DR GO; GO:0050660; F:flavin adenine dinucleotide binding; IDA:UniProtKB.
DR GO; GO:0043640; P:benzoate catabolic process via hydroxylation; IEA:UniProtKB-UniPathway.
DR Gene3D; 3.50.50.60; -; 1.
DR InterPro; IPR002938; FAD-bd.
DR InterPro; IPR036188; FAD/NAD-bd_sf.
DR InterPro; IPR012733; HB_mOase.
DR Pfam; PF01494; FAD_binding_3; 1.
DR SUPFAM; SSF51905; SSF51905; 1.
DR TIGRFAMs; TIGR02360; pbenz_hydroxyl; 1.
PE 1: Evidence at protein level;
KW 3D-structure; Aromatic hydrocarbons catabolism; Direct protein sequencing;
KW FAD; Flavoprotein; Monooxygenase; NADP; Oxidoreductase.
FT CHAIN 1..394
FT /note="p-hydroxybenzoate hydroxylase"
FT /id="PRO_0000058382"
FT BINDING 13
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 32
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 42..47
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 102
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 201
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 212..214
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 222
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 286
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 293
FT /ligand="substrate"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT BINDING 299..300
FT /ligand="FAD"
FT /ligand_id="ChEBI:CHEBI:57692"
FT /evidence="ECO:0000269|PubMed:10025942,
FT ECO:0000269|PubMed:10493859, ECO:0000269|PubMed:2553983,
FT ECO:0000269|PubMed:2819062, ECO:0000269|PubMed:3351945,
FT ECO:0000269|PubMed:7520279, ECO:0000269|PubMed:7628466,
FT ECO:0000269|PubMed:7756982, ECO:0000269|PubMed:9578477,
FT ECO:0000269|PubMed:9694855"
FT SITE 201
FT /note="Important for catalytic activity"
FT /evidence="ECO:0000250|UniProtKB:P20586"
FT SITE 385
FT /note="Important for catalytic activity"
FT /evidence="ECO:0000250|UniProtKB:P20586"
FT MUTAGEN 33
FT /note="R->E: Slight decrease of affinity for p-OHB and
FT strong decrease of affinity for NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 33
FT /note="R->K: Slight decrease of affinity for p-OHB and
FT NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 33
FT /note="R->S: Slight decrease of affinity for p-OHB and
FT strong decrease of affinity for NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 34
FT /note="Q->K: Slight decrease of affinity for p-OHB and
FT NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 34
FT /note="Q->R: Slight decrease of affinity for p-OHB and
FT NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 34
FT /note="Q->T: Slight decrease of affinity for p-OHB and
FT NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 38
FT /note="Y->E: Slight decrease of affinity for p-OHB and
FT strong decrease of affinity for NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 38
FT /note="Y->F: Slight decrease of affinity for p-OHB and
FT strong decrease of affinity for NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 38
FT /note="Y->K: Slight decrease of affinity for p-OHB and
FT strong decrease of affinity for NADPH."
FT /evidence="ECO:0000269|PubMed:10493859"
FT MUTAGEN 42
FT /note="R->K: 4-fold and 10-fold decrease of affinity for p-
FT OHB and NADPH, respectively. The turnover rate of p-
FT hydroxybenzoate hydroxylase results from impaired binding
FT of NADPH."
FT /evidence="ECO:0000269|PubMed:9578477"
FT MUTAGEN 42
FT /note="R->S: 3-fold and 10-fold decrease of affinity for p-
FT OHB and NADPH, respectively. The turnover rate of p-
FT hydroxybenzoate hydroxylase results from impaired binding
FT of NADPH. Hardly disturbs the binding of FAD."
FT /evidence="ECO:0000269|PubMed:9578477"
FT MUTAGEN 44
FT /note="R->K: Decrease of affinity for the flavin prosthetic
FT group. It affects NADPH binding, resulting in a low yield
FT of the charge-transfer species between reduced flavin and
FT NADP."
FT /evidence="ECO:0000269|PubMed:7628466"
FT MUTAGEN 116
FT /note="C->S: Slight decrease of affinity for NADPH and p-
FT OHB are observed."
FT /evidence="ECO:0000269|PubMed:1459126"
FT MUTAGEN 161
FT /note="F->A: Decrease of affinity for NADPH."
FT /evidence="ECO:0000269|PubMed:10025942"
FT MUTAGEN 161
FT /note="F->G: Decrease of affinity for NADPH."
FT /evidence="ECO:0000269|PubMed:10025942"
FT MUTAGEN 162
FT /note="H->D: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 162
FT /note="H->K: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT slightly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 162
FT /note="H->N: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 162
FT /note="H->R: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT slightly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 162
FT /note="H->S: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 162
FT /note="H->T: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 162
FT /note="H->Y: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT slightly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 166
FT /note="R->E: Loses the ability to bind NADPH and FAD."
FT /evidence="ECO:0000269|PubMed:10025942"
FT MUTAGEN 166
FT /note="R->K: Loses the ability to bind NADPH."
FT /evidence="ECO:0000269|PubMed:10025942"
FT MUTAGEN 166
FT /note="R->S: Loses the ability to bind NADPH."
FT /evidence="ECO:0000269|PubMed:10025942"
FT MUTAGEN 214
FT /note="R->K: Strong decrease of affinity for NADPH and 4-
FT fold decrease of affinity for p-OHB are observed."
FT /evidence="ECO:0000269|PubMed:1459126"
FT MUTAGEN 222
FT /note="Y->A: Results in the removal of a large side chain
FT involving in the binding of the carboxyl group of the
FT substrate."
FT /evidence="ECO:0000269|PubMed:7520279"
FT MUTAGEN 269
FT /note="R->D: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 269
FT /note="R->K: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT slightly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 269
FT /note="R->N: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 269
FT /note="R->S: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT slightly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 269
FT /note="R->T: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT MUTAGEN 269
FT /note="R->Y: No significant changes in affinity for p-OHB
FT are observed. However, the affinity for NADPH decreases
FT strongly."
FT /evidence="ECO:0000269|PubMed:9694855"
FT CONFLICT 344
FT /note="W -> Y (in Ref. 2; AA sequence)"
FT /evidence="ECO:0000305"
FT STRAND 4..8
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 12..23
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 28..31
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 36..40
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 47..49
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 50..58
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 63..68
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 70..79
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 82..87
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 88..91
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 97..99
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 102..116
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 119..121
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 125..130
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 134..136
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 138..143
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 146..151
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 153..157
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 164..167
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 171..173
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 175..192
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 195..198
FT /evidence="ECO:0007829|PDB:1BGN"
FT STRAND 200..203
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 209..215
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 218..225
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 231..233
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 236..246
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 249..254
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 260..278
FT /evidence="ECO:0007829|PDB:1PBE"
FT STRAND 281..283
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 285..287
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 293..295
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 298..318
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 322..327
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 328..350
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 358..371
FT /evidence="ECO:0007829|PDB:1PBE"
FT HELIX 375..386
FT /evidence="ECO:0007829|PDB:1PBE"
SQ SEQUENCE 394 AA; 44322 MW; D29599224AC81E00 CRC64;
MKTQVAIIGA GPSGLLLGQL LHKAGIDNVI LERQTPDYVL GRIRAGVLEQ GMVDLLREAG
VDRRMARDGL VHEGVEIAFA GQRRRIDLKR LSGGKTVTVY GQTEVTRDLM EAREACGATT
VYQAAEVRLH DLQGERPYVT FERDGERLRL DCDYIAGCDG FHGISRQSIP AERLKVFERV
YPFGWLGLLA DTPPVSHELI YANHPRGFAL CSQRSATRSR YYVQVPLTEK VEDWSDERFW
TELKARLPAE VAEKLVTGPS LEKSIAPLRS FVVEPMQHGR LFLAGDAAHI VPPTGAKGLN
LAASDVSTLY RLLLKAYREG RGELLERYSA ICLRRIWKAE RFSWWMTSVL HRFPDTDAFS
QRIQQTELEY YLGSEAGLAT IAENYVGLPY EEIE
