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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

Manual assertion based on experiment ini

  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)DescriptionActionsGraphical viewLength
    Binding sitei13FAD10 Publications1
    Binding sitei32FAD10 Publications1
    Binding sitei102FAD10 Publications1
    Binding sitei201Substrate10 Publications1
    Sitei201Important for catalytic activityBy similarity1
    Binding sitei222Substrate10 Publications1
    Binding sitei286FAD10 Publications1
    Binding sitei293Substrate; via carbonyl oxygen10 Publications1
    Sitei385Important for catalytic activityBy similarity1

    Regions

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Nucleotide bindingi42 – 47FAD10 Publications6
    Nucleotide bindingi299 – 300FAD10 Publications2

    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.
    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)DescriptionActionsGraphical viewLength
    Mutagenesisi33R → E: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication1
    Mutagenesisi33R → K: Slight decrease of affinity for p-OHB and NADPH. 1 Publication1
    Mutagenesisi33R → S: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication1
    Mutagenesisi34Q → K: Slight decrease of affinity for p-OHB and NADPH. 1 Publication1
    Mutagenesisi34Q → R: Slight decrease of affinity for p-OHB and NADPH. 1 Publication1
    Mutagenesisi34Q → T: Slight decrease of affinity for p-OHB and NADPH. 1 Publication1
    Mutagenesisi38Y → E: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication1
    Mutagenesisi38Y → F: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication1
    Mutagenesisi38Y → K: Slight decrease of affinity for p-OHB and strong decrease of affinity for NADPH. 1 Publication1
    Mutagenesisi42R → 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 Publication1
    Mutagenesisi42R → 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 Publication1
    Mutagenesisi44R → 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 Publication1
    Mutagenesisi116C → S: Slight decrease of affinity for NADPH and p-OHB are observed. 1 Publication1
    Mutagenesisi161F → A: Decrease of affinity for NADPH. 1 Publication1
    Mutagenesisi161F → G: Decrease of affinity for NADPH. 1 Publication1
    Mutagenesisi162H → D: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1
    Mutagenesisi162H → K: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication1
    Mutagenesisi162H → N: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1
    Mutagenesisi162H → R: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication1
    Mutagenesisi162H → S: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1
    Mutagenesisi162H → T: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1
    Mutagenesisi162H → Y: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication1
    Mutagenesisi166R → E: Loses the ability to bind NADPH and FAD. 1 Publication1
    Mutagenesisi166R → K: Loses the ability to bind NADPH. 1 Publication1
    Mutagenesisi166R → S: Loses the ability to bind NADPH. 1 Publication1
    Mutagenesisi214R → K: Strong decrease of affinity for NADPH and 4-fold decrease of affinity for p-OHB are observed. 1 Publication1
    Mutagenesisi222Y → A: Results in the removal of a large side chain involving in the binding of the carboxyl group of the substrate. 1 Publication1
    Mutagenesisi269R → D: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1
    Mutagenesisi269R → K: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication1
    Mutagenesisi269R → N: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1
    Mutagenesisi269R → S: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases slightly. 1 Publication1
    Mutagenesisi269R → T: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1
    Mutagenesisi269R → Y: No significant changes in affinity for p-OHB are observed. However, the affinity for NADPH decreases strongly. 1 Publication1

    Chemistry databases

    ChEMBLiCHEMBL3675.
    DrugBankiDB03147. Flavin adenine dinucleotide.

    PTM / Processingi

    Molecule processing

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    ChainiPRO_00000583821 – 394p-hydroxybenzoate hydroxylaseAdd BLAST394

    Interactioni

    Subunit structurei

    Homodimer.11 Publications

    Chemistry databases

    BindingDBiP00438.

    Structurei

    Secondary structure

    1394
    Legend: HelixTurnBeta strandPDB Structure known for this area
    Show more details
    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Beta strandi4 – 8Combined sources5
    Helixi12 – 23Combined sources12
    Beta strandi28 – 31Combined sources4
    Helixi36 – 40Combined sources5
    Beta strandi47 – 49Combined sources3
    Helixi50 – 58Combined sources9
    Helixi63 – 68Combined sources6
    Beta strandi70 – 79Combined sources10
    Beta strandi82 – 87Combined sources6
    Helixi88 – 91Combined sources4
    Beta strandi97 – 99Combined sources3
    Helixi102 – 116Combined sources15
    Beta strandi119 – 121Combined sources3
    Beta strandi125 – 130Combined sources6
    Beta strandi134 – 136Combined sources3
    Beta strandi138 – 143Combined sources6
    Beta strandi146 – 151Combined sources6
    Beta strandi153 – 157Combined sources5
    Helixi164 – 167Combined sources4
    Helixi171 – 173Combined sources3
    Beta strandi175 – 192Combined sources18
    Beta strandi195 – 198Combined sources4
    Beta strandi200 – 203Combined sources4
    Beta strandi209 – 215Combined sources7
    Beta strandi218 – 225Combined sources8
    Helixi231 – 233Combined sources3
    Helixi236 – 246Combined sources11
    Helixi249 – 254Combined sources6
    Beta strandi260 – 278Combined sources19
    Beta strandi281 – 283Combined sources3
    Helixi285 – 287Combined sources3
    Helixi293 – 295Combined sources3
    Helixi298 – 318Combined sources21
    Helixi322 – 327Combined sources6
    Helixi328 – 350Combined sources23
    Helixi358 – 371Combined sources14
    Helixi375 – 386Combined sources12

    3D structure databases

    Select the link destinations:
    PDBei
    RCSB PDBi
    PDBji
    Links Updated
    PDB 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.
    ModBaseiSearch...
    MobiDBiSearch...

    Miscellaneous databases

    EvolutionaryTraceiP00438.

    Family & Domainsi

    Region

    Feature keyPosition(s)DescriptionActionsGraphical viewLength
    Regioni212 – 214Substrate binding10 Publications3

    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)DescriptionActionsGraphical viewLength
    Sequence conflicti344W → Y AA sequence (PubMed:6809053).Curated1

    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
    PDB 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.
    ModBaseiSearch...
    MobiDBiSearch...

    Chemistry databases

    BindingDBiP00438.
    ChEMBLiCHEMBL3675.
    DrugBankiDB03147. Flavin adenine dinucleotide.

    Protocols and materials databases

    Structural Biology KnowledgebaseSearch...

    Enzyme and pathway databases

    UniPathwayiUPA00156; UER00257.
    BioCyciMetaCyc:MONOMER-11534.
    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: November 2, 2016
    This is version 112 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.