PDBsum entry 1bgn

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Oxidoreductase PDB id
Protein chain
391 a.a. *
Waters ×279
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: P-hydroxybenzoate hydroxylase (phbh) mutant with cys 116 rep ser (c116s) and arg 269 replaced by thr (r269t), in complex and 4-hydroxybenzoic acid
Structure: P-hydroxybenzoate hydroxylase. Chain: a. Engineered: yes. Mutation: yes
Source: Pseudomonas fluorescens. Organism_taxid: 294. Gene: poba. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PQS)
2.00Å     R-factor:   0.177    
Authors: M.H.M.Eppink,H.A.Schreuder,W.J.H.Van Berkel
Key ref:
M.H.Eppink et al. (1998). Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested by kinetic, crystallographic and modeling studies of histidine 162 and arginine 269 variants. J Biol Chem, 273, 21031-21039. PubMed id: 9694855 DOI: 10.1074/jbc.273.33.21031
29-May-98     Release date:   12-Aug-98    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P00438  (PHHY_PSEFL) -  p-hydroxybenzoate hydroxylase
394 a.a.
391 a.a.*
Key:    PfamA domain  Secondary structure  CATH domain
* PDB and UniProt seqs differ at 2 residue positions (black crosses)

 Enzyme reactions 
   Enzyme class: E.C.  - 4-hydroxybenzoate 3-monooxygenase.
[IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Benzoate Metabolism
      Reaction: 4-hydroxybenzoate + NADPH + O2 = protocatechuate + NADP+ + H2O
Bound ligand (Het Group name = PHB)
corresponds exactly
+ O(2)
= protocatechuate
+ NADP(+)
+ H(2)O
      Cofactor: FAD
Bound ligand (Het Group name = FAD) corresponds exactly
Molecule diagrams generated from .mol files obtained from the KEGG ftp site
 Gene Ontology (GO) functional annotation 
  GO annot!
  Biological process     metabolic process   5 terms 
  Biochemical function     oxidoreductase activity     4 terms  


DOI no: 10.1074/jbc.273.33.21031 J Biol Chem 273:21031-21039 (1998)
PubMed id: 9694855  
Interdomain binding of NADPH in p-hydroxybenzoate hydroxylase as suggested by kinetic, crystallographic and modeling studies of histidine 162 and arginine 269 variants.
M.H.Eppink, H.A.Schreuder, W.J.van Berkel.
The conserved residues His-162 and Arg-269 of the flavoprotein p-hydroxybenzoate hydroxylase (EC are located at the entrance of the interdomain cleft that leads toward the active site. To study their putative role in NADPH binding, His-162 and Arg-269 were selectively changed by site-specific mutagenesis. The catalytic properties of H162R, H162Y, and R269K were similar to the wild-type enzyme. However, less conservative His-162 and Arg-269 replacements strongly impaired NADPH binding without affecting the conformation of the flavin ring and the efficiency of substrate hydroxylation. The crystal structures of H162R and R269T in complex with 4-hydroxybenzoate were solved at 3.0 and 2.0 A resolution, respectively. Both structures are virtually indistinguishable from the wild-type enzyme-substrate complex except for the substituted side chains. In contrast to wild-type p-hydroxybenzoate hydroxylase, H162R is not inactivated by diethyl pyrocarbonate. NADPH protects wild-type p-hydroxybenzoate hydroxylase from diethylpyrocarbonate inactivation, suggesting that His-162 is involved in NADPH binding. Based on these results and GRID calculations we propose that the side chains of His-162 and Arg-269 interact with the pyrophosphate moiety of NADPH. An interdomain binding mode for NADPH is proposed which takes a novel sequence motif (Eppink, M. H. M., Schreuder, H. A., and van Berkel, W. J. H. (1997) Protein Sci. 6, 2454-2458) into account.
  Selected figure(s)  
Figure 1.
Fig. 1. Domain structure of p-hydroxybenzoate hydroxylase. The FAD domain (orange), substrate domain (green), and interface domain (blue) are indicated. The substrate (POHB) is in red and the FAD in yellow. The amino acid residues His-162 and Arg-269 are depicted in magenta. Data were taken from the crystal structure of the enzyme-substrate complex refined at 1.9 Å resolution (10).
Figure 6.
Fig. 6. Stereo drawing of an F[o]-F[c] omit map of H162R, contoured at 3 . The atomic model of H162R with residue Arg-162 is drawn in black bonds, while the orientation of His-162 in wtPHBH is drawn in open bonds.
  The above figures are reprinted by permission from the ASBMB: J Biol Chem (1998, 273, 21031-21039) copyright 1998.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19060149 S.D.Pope, L.L.Chen, and V.Stewart (2009).
Purine utilization by Klebsiella oxytoca M5al: genes for ring-oxidizing and -opening enzymes.
  J Bacteriol, 191, 1006-1017.  
18176933 E.V.Kudryashova, A.J.Visser, and W.J.van Berkel (2008).
Monomer formation and function of p-hydroxybenzoate hydroxylase in reverse micelles and in dimethylsulfoxide/water mixtures.
  Chembiochem, 9, 413-419.  
16275925 C.Siebold, N.Berrow, T.S.Walter, K.Harlos, R.J.Owens, D.I.Stuart, J.R.Terman, A.L.Kolodkin, R.J.Pasterkamp, and E.Y.Jones (2005).
High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule.
  Proc Natl Acad Sci U S A, 102, 16836-16841.
PDB codes: 2bry 2c4c
16267294 X.Gao, C.L.Tan, C.C.Yeo, and C.L.Poh (2005).
Molecular and biochemical characterization of the xlnD-encoded 3-hydroxybenzoate 6-hydroxylase involved in the degradation of 2,5-xylenol via the gentisate pathway in Pseudomonas alcaligenes NCIMB 9867.
  J Bacteriol, 187, 7696-7702.  
12081493 B.A.Palfey, R.Basu, K.K.Frederick, B.Entsch, and D.P.Ballou (2002).
Role of protein flexibility in the catalytic cycle of p-hydroxybenzoate hydroxylase elucidated by the Pro293Ser mutant.
  Biochemistry, 41, 8438-8446.  
11805318 J.Wang, M.Ortiz-Maldonado, B.Entsch, V.Massey, D.Ballou, and D.L.Gatti (2002).
Protein and ligand dynamics in 4-hydroxybenzoate hydroxylase.
  Proc Natl Acad Sci U S A, 99, 608-613.
PDB codes: 1k0i 1k0j 1k0l
11082194 M.H.Eppink, E.Cammaart, D.Van Wassenaar, W.J.Middelhoven, and W.J.van Berkel (2000).
Purification and properties of hydroquinone hydroxylase, a FAD-dependent monooxygenase involved in the catabolism of 4-hydroxybenzoate in Candida parapsilosis CBS604.
  Eur J Biochem, 267, 6832-6840.  
10600126 M.Ortiz-Maldonado, D.Gatti, D.P.Ballou, and V.Massey (1999).
Structure-function correlations of the reaction of reduced nicotinamide analogues with p-hydroxybenzoate hydroxylase substituted with a series of 8-substituted flavins.
  Biochemistry, 38, 16636-16647.
PDB code: 1d7l
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