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Protein

p-hydroxybenzoate hydroxylase

Gene

pobA

Organism
Pseudomonas fluorescens
Status
Reviewed-Annotation score: Annotation score: 5 out of 5-Experimental evidence at protein leveli

Functioni

Catalyzes the incorporation of an atom of dioxygen into p-hydroxybenzoate (p-OHB) to form 3,4-dihydroxybenzoate (3,4DOHB). The reaction occurs in two parts: reduction of the flavin adenine dinucleotide (FAD) in the enzyme by reduced nicotinamide adenine dinucleotide phosphate (NADPH) in response to binding p-hydroxybenzoate to the enzyme and oxidation of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate.10 Publications

Catalytic activityi

4-hydroxybenzoate + NADPH + O2 = protocatechuate + NADP+ + H2O.7 Publications

Cofactori

FAD9 PublicationsNote: Binds 1 FAD per subunit.7 Publications

Kineticsi

Kcat is 55 sec(-1) for hydroxylase activity (PubMed:9578477, PubMed:9694855). Kcat is 55 sec(-1) for hydroxylase activity (at 25 degrees Celsius) (PubMed:1459126). Kcat is 55 sec(-1) for hydroxylase activity (at pH 8) (PubMed:10025942, PubMed:10493859). Kcat is 9 sec(-1) for hydroxylase activity (at 6 degrees Celsius) (PubMed:1459126).6 Publications

  1. KM=15 µM for p-OHB1 Publication
  2. KM=15 µM for p-OHB (at pH 6)2 Publications
  3. KM=20 µM for p-OHB2 Publications
  4. KM=25 µM for p-OHB (at 25 degrees Celsius)1 Publication
  5. KM=30 µM for NADPH1 Publication
  6. KM=30 µM for p-OHB (at 6 degrees Celsius)1 Publication
  7. KM=34 µM for NADPH (at pH 6)2 Publications
  8. KM=40 µM for NADPH (at 6 degrees Celsius)1 Publication
  9. KM=50 µM for NADPH (at 25 degrees Celsius)1 Publication
  10. KM=50 µM for NADPH1 Publication
  11. KM=70 µM for NADPH1 Publication

    Pathwayi: benzoate degradation via hydroxylation

    This protein is involved in step 2 of the subpathway that synthesizes 3,4-dihydroxybenzoate from benzoate.Curated
    Proteins known to be involved in the 2 steps of the subpathway in this organism are:
    1. no protein annotated in this organism
    2. p-hydroxybenzoate hydroxylase (pobA)
    This subpathway is part of the pathway benzoate degradation via hydroxylation, which is itself part of Aromatic compound metabolism.
    View all proteins of this organism that are known to be involved in the subpathway that synthesizes 3,4-dihydroxybenzoate from benzoate, the pathway benzoate degradation via hydroxylation and in Aromatic compound metabolism.

    Sites

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Binding sitei13 – 131FAD10 Publications
    Binding sitei32 – 321FAD10 Publications
    Binding sitei102 – 1021FAD10 Publications
    Binding sitei201 – 2011Substrate10 Publications
    Sitei201 – 2011Important for catalytic activityBy similarity
    Binding sitei222 – 2221Substrate10 Publications
    Binding sitei286 – 2861FAD10 Publications
    Binding sitei293 – 2931Substrate; via carbonyl oxygen10 Publications
    Sitei385 – 3851Important for catalytic activityBy similarity

    Regions

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Nucleotide bindingi42 – 476FAD10 Publications
    Nucleotide bindingi299 – 3002FAD10 Publications

    GO - Molecular functioni

    • 4-hydroxybenzoate 3-monooxygenase activity Source: UniProtKB
    • FAD binding Source: UniProtKB
    • flavin adenine dinucleotide binding Source: UniProtKB

    GO - Biological processi

    Complete GO annotation...

    Keywords - Molecular functioni

    Monooxygenase, Oxidoreductase

    Keywords - Biological processi

    Aromatic hydrocarbons catabolism

    Keywords - Ligandi

    FAD, Flavoprotein, NADP

    Enzyme and pathway databases

    BioCyciMetaCyc:MONOMER-11534.
    RETL1328306-WGS:GSTH-3505-MONOMER.
    BRENDAi1.14.13.2. 5121.
    UniPathwayiUPA00156; UER00257.

    Names & Taxonomyi

    Protein namesi
    Recommended name:
    p-hydroxybenzoate hydroxylase1 Publication (EC:1.14.13.27 Publications)
    Short name:
    PHBH1 Publication
    Short name:
    PHBHase1 Publication
    Alternative name(s):
    4-hydroxybenzoate 3-monooxygenaseCurated
    Gene namesi
    Name:pobA
    OrganismiPseudomonas fluorescens
    Taxonomic identifieri294 [NCBI]
    Taxonomic lineageiBacteriaProteobacteriaGammaproteobacteriaPseudomonadalesPseudomonadaceaePseudomonas

    Pathology & Biotechi

    Mutagenesis

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Mutagenesisi33 – 331R → E: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication
    Mutagenesisi33 – 331R → K: Slight decrease of affinity for p-OHB and NADPH. 1 Publication
    Mutagenesisi33 – 331R → S: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication
    Mutagenesisi34 – 341Q → K: Slight decrease of affinity for p-OHB and NADPH. 1 Publication
    Mutagenesisi34 – 341Q → R: Slight decrease of affinity for p-OHB and NADPH. 1 Publication
    Mutagenesisi34 – 341Q → T: Slight decrease of affinity for p-OHB and NADPH. 1 Publication
    Mutagenesisi38 – 381Y → E: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication
    Mutagenesisi38 – 381Y → F: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication
    Mutagenesisi38 – 381Y → K: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication
    Mutagenesisi42 – 421R → K: 4-fold and 10-fold decrease of affinity for p-OHB and NADPH, respectively. The turnover rate of p-hydroxybenzoate hydroxylase results from impaired binding of NADPH. 1 Publication
    Mutagenesisi42 – 421R → S: 3-fold and 10-fold decrease of affinity for p-OHB and NADPH, respectively. The turnover rate of p-hydroxybenzoate hydroxylase results from impaired binding of NADPH. Hardly disturbs the binding of FAD. 1 Publication
    Mutagenesisi44 – 441R → K: Decrease of affinity for the flavin prosthetic group. It affects NADPH binding, resulting in a low yield of the charge-transfer species between reduced flavin and NADP. 1 Publication
    Mutagenesisi116 – 1161C → S: Slight decrease of affinity for NADPH and p-OHB are observed. 1 Publication
    Mutagenesisi161 – 1611F → A: Decrease of affinity for NADPH. 1 Publication
    Mutagenesisi161 – 1611F → G: Decrease of affinity for NADPH. 1 Publication
    Mutagenesisi162 – 1621H → D: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication
    Mutagenesisi162 – 1621H → K: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication
    Mutagenesisi162 – 1621H → N: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication
    Mutagenesisi162 – 1621H → R: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication
    Mutagenesisi162 – 1621H → S: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication
    Mutagenesisi162 – 1621H → T: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication
    Mutagenesisi162 – 1621H → Y: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication
    Mutagenesisi166 – 1661R → E: Loses the ability to bind NADPH and FAD. 1 Publication
    Mutagenesisi166 – 1661R → K: Loses the ability to bind NADPH. 1 Publication
    Mutagenesisi166 – 1661R → S: Loses the ability to bind NADPH. 1 Publication
    Mutagenesisi214 – 2141R → K: Strong decrease of affinity for NADPH and 4-fold decrease of affinity for p-OHB are observed. 1 Publication
    Mutagenesisi222 – 2221Y → A: Results in the removal of a large side chain involving in the binding of the carboxyl group of the substrate. 1 Publication
    Mutagenesisi269 – 2691R → D: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication
    Mutagenesisi269 – 2691R → K: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication
    Mutagenesisi269 – 2691R → N: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication
    Mutagenesisi269 – 2691R → S: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication
    Mutagenesisi269 – 2691R → T: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication
    Mutagenesisi269 – 2691R → Y: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication

    Chemistry

    ChEMBLiCHEMBL3675.
    DrugBankiDB03147. Flavin adenine dinucleotide.

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Chaini1 – 394394p-hydroxybenzoate hydroxylasePRO_0000058382Add
    BLAST

    Interactioni

    Subunit structurei

    Homodimer.11 Publications

    Chemistry

    BindingDBiP00438.

    Structurei

    Secondary structure

    1
    394
    Legend: HelixTurnBeta strand
    Show more details
    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Beta strandi4 – 85Combined sources
    Helixi12 – 2312Combined sources
    Beta strandi28 – 314Combined sources
    Helixi36 – 405Combined sources
    Beta strandi47 – 493Combined sources
    Helixi50 – 589Combined sources
    Helixi63 – 686Combined sources
    Beta strandi70 – 7910Combined sources
    Beta strandi82 – 876Combined sources
    Helixi88 – 914Combined sources
    Beta strandi97 – 993Combined sources
    Helixi102 – 11615Combined sources
    Beta strandi119 – 1213Combined sources
    Beta strandi125 – 1306Combined sources
    Beta strandi134 – 1363Combined sources
    Beta strandi138 – 1436Combined sources
    Beta strandi146 – 1516Combined sources
    Beta strandi153 – 1575Combined sources
    Helixi164 – 1674Combined sources
    Helixi171 – 1733Combined sources
    Beta strandi175 – 19218Combined sources
    Beta strandi195 – 1984Combined sources
    Beta strandi200 – 2034Combined sources
    Beta strandi209 – 2157Combined sources
    Beta strandi218 – 2258Combined sources
    Helixi231 – 2333Combined sources
    Helixi236 – 24611Combined sources
    Helixi249 – 2546Combined sources
    Beta strandi260 – 27819Combined sources
    Beta strandi281 – 2833Combined sources
    Helixi285 – 2873Combined sources
    Helixi293 – 2953Combined sources
    Helixi298 – 31821Combined sources
    Helixi322 – 3276Combined sources
    Helixi328 – 35023Combined sources
    Helixi358 – 37114Combined sources
    Helixi375 – 38612Combined sources

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1BF3X-ray2.20A1-394[»]
    1BGJX-ray3.00A1-394[»]
    1BGNX-ray2.00A1-394[»]
    1BKWX-ray2.20A1-394[»]
    1CC4X-ray2.00A1-394[»]
    1CC6X-ray2.20A1-394[»]
    1CJ2X-ray2.80A1-391[»]
    1CJ3X-ray2.50A1-392[»]
    1CJ4X-ray2.40A1-392[»]
    1PBBX-ray2.50A1-394[»]
    1PBCX-ray2.80A1-394[»]
    1PBDX-ray2.30A1-394[»]
    1PBEX-ray1.90A1-394[»]
    1PBFX-ray2.70A1-394[»]
    1PDHX-ray2.10A1-394[»]
    1PHHX-ray2.30A1-394[»]
    2PHHX-ray2.70A1-394[»]
    ProteinModelPortaliP00438.
    SMRiP00438. Positions 1-394.
    ModBaseiSearch...
    MobiDBiSearch...

    Miscellaneous databases

    EvolutionaryTraceiP00438.

    Family & Domainsi

    Region

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Regioni212 – 2143Substrate binding10 Publications

    Sequence similaritiesi

    Belongs to the aromatic-ring hydroxylase family.Curated

    Family and domain databases

    Gene3Di3.50.50.60. 1 hit.
    InterProiIPR002938. FAD-bd.
    IPR023753. FAD/NAD-binding_dom.
    IPR012733. HB_mOase.
    [Graphical view]
    PfamiPF01494. FAD_binding_3. 1 hit.
    [Graphical view]
    SUPFAMiSSF51905. SSF51905. 2 hits.
    TIGRFAMsiTIGR02360. pbenz_hydroxyl. 1 hit.

    Sequencei

    Sequence statusi: Complete.

    P00438-1 [UniParc]FASTAAdd to basket

    « Hide

            10         20         30         40         50
    MKTQVAIIGA GPSGLLLGQL LHKAGIDNVI LERQTPDYVL GRIRAGVLEQ
    60 70 80 90 100
    GMVDLLREAG VDRRMARDGL VHEGVEIAFA GQRRRIDLKR LSGGKTVTVY
    110 120 130 140 150
    GQTEVTRDLM EAREACGATT VYQAAEVRLH DLQGERPYVT FERDGERLRL
    160 170 180 190 200
    DCDYIAGCDG FHGISRQSIP AERLKVFERV YPFGWLGLLA DTPPVSHELI
    210 220 230 240 250
    YANHPRGFAL CSQRSATRSR YYVQVPLTEK VEDWSDERFW TELKARLPAE
    260 270 280 290 300
    VAEKLVTGPS LEKSIAPLRS FVVEPMQHGR LFLAGDAAHI VPPTGAKGLN
    310 320 330 340 350
    LAASDVSTLY RLLLKAYREG RGELLERYSA ICLRRIWKAE RFSWWMTSVL
    360 370 380 390
    HRFPDTDAFS QRIQQTELEY YLGSEAGLAT IAENYVGLPY EEIE
    Length:394
    Mass (Da):44,322
    Last modified:April 1, 1993 - v2
    Checksum:iD29599224AC81E00
    GO

    Experimental Info

    Feature keyPosition(s)LengthDescriptionGraphical viewFeature identifierActions
    Sequence conflicti344 – 3441W → Y AA sequence (PubMed:6809053).Curated

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    X68438 Genomic DNA. Translation: CAA48483.1.
    PIRiA90643. WHPSBF.

    Cross-referencesi

    Sequence databases

    Select the link destinations:
    EMBLi
    GenBanki
    DDBJi
    Links Updated
    X68438 Genomic DNA. Translation: CAA48483.1.
    PIRiA90643. WHPSBF.

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    EntryMethodResolution (Å)ChainPositionsPDBsum
    1BF3X-ray2.20A1-394[»]
    1BGJX-ray3.00A1-394[»]
    1BGNX-ray2.00A1-394[»]
    1BKWX-ray2.20A1-394[»]
    1CC4X-ray2.00A1-394[»]
    1CC6X-ray2.20A1-394[»]
    1CJ2X-ray2.80A1-391[»]
    1CJ3X-ray2.50A1-392[»]
    1CJ4X-ray2.40A1-392[»]
    1PBBX-ray2.50A1-394[»]
    1PBCX-ray2.80A1-394[»]
    1PBDX-ray2.30A1-394[»]
    1PBEX-ray1.90A1-394[»]
    1PBFX-ray2.70A1-394[»]
    1PDHX-ray2.10A1-394[»]
    1PHHX-ray2.30A1-394[»]
    2PHHX-ray2.70A1-394[»]
    ProteinModelPortaliP00438.
    SMRiP00438. Positions 1-394.
    ModBaseiSearch...
    MobiDBiSearch...

    Chemistry

    BindingDBiP00438.
    ChEMBLiCHEMBL3675.
    DrugBankiDB03147. Flavin adenine dinucleotide.

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Enzyme and pathway databases

    UniPathwayiUPA00156; UER00257.
    BioCyciMetaCyc:MONOMER-11534.
    RETL1328306-WGS:GSTH-3505-MONOMER.
    BRENDAi1.14.13.2. 5121.

    Miscellaneous databases

    EvolutionaryTraceiP00438.

    Family and domain databases

    Gene3Di3.50.50.60. 1 hit.
    InterProiIPR002938. FAD-bd.
    IPR023753. FAD/NAD-binding_dom.
    IPR012733. HB_mOase.
    [Graphical view]
    PfamiPF01494. FAD_binding_3. 1 hit.
    [Graphical view]
    SUPFAMiSSF51905. SSF51905. 2 hits.
    TIGRFAMsiTIGR02360. pbenz_hydroxyl. 1 hit.
    ProtoNetiSearch...

    Entry informationi

    Entry nameiPHHY_PSEFL
    AccessioniPrimary (citable) accession number: P00438
    Entry historyi
    Integrated into UniProtKB/Swiss-Prot: July 21, 1986
    Last sequence update: April 1, 1993
    Last modified: May 11, 2016
    This is version 110 of the entry and version 2 of the sequence. [Complete history]
    Entry statusiReviewed (UniProtKB/Swiss-Prot)
    Annotation programProkaryotic Protein Annotation Program

    Miscellaneousi

    Keywords - Technical termi

    3D-structure, Direct protein sequencing

    Documents

    1. PATHWAY comments
      Index of metabolic and biosynthesis pathways
    2. PDB cross-references
      Index of Protein Data Bank (PDB) cross-references
    3. SIMILARITY comments
      Index of protein domains and families

    Similar proteinsi

    Links to similar proteins from the UniProt Reference Clusters (UniRef) at 100%, 90% and 50% sequence identity:
    100%UniRef100 combines identical sequences and sub-fragments with 11 or more residues from any organism into one UniRef entry.
    90%UniRef90 is built by clustering UniRef100 sequences that have at least 90% sequence identity to, and 80% overlap with, the longest sequence (a.k.a seed sequence).
    50%UniRef50 is built by clustering UniRef90 seed sequences that have at least 50% sequence identity to, and 80% overlap with, the longest sequence in the cluster.