PDBsum entry 1bf3

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protein ligands links
Oxidoreductase PDB id
Protein chain
391 a.a. *
Waters ×289
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: P-hydroxybenzoate hydroxylase (phbh) mutant with cys 116 rep ser (c116s) and arg 42 replaced by lys (r42k), in complex w 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)
2.20Å     R-factor:   0.175    
Authors: M.H.M.Eppink,H.A.Schreuder,W.J.H.Van Berkel
Key ref: M.H.Eppink et al. (1998). Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding. Eur J Biochem, 253, 194-201. PubMed id: 9578477
26-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  


Eur J Biochem 253:194-201 (1998)
PubMed id: 9578477  
Lys42 and Ser42 variants of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens reveal that Arg42 is essential for NADPH binding.
M.H.Eppink, H.A.Schreuder, W.J.van Berkel.
The conserved Arg42 of the flavoprotein p-hydroxybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens by more than two orders of magnitude. Rapid reaction kinetics show that the low activity of the Arg42 variants results from impaired binding of NADPH. In contrast to an earlier conclusion drawn for p-hydroxybenzoate hydroxylase from Acinetobacter calcoaceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluorescens hardly disturbs the binding of FAD. Crystals of [Lys42]p-hydroxybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.22-nm resolution. The structure of the Lys42 variant is virtually indistinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structure interacts indirectly via a solvent molecule with the 3-OH of the adenosine ribose moiety of FAD. Substrate perturbation difference spectra suggest that the Arg42 replacements influence the solvent accessibility of the flavin ring in the oxidized enzyme. In spite of this, the Arg42 variants fully couple enzyme reduction to substrate hydroxylation. Sequence-comparison studies suggest that Arg42 is involved in binding of the 2'-phosphoadenosine moiety of NADPH.

Literature references that cite this PDB file's key reference

  PubMed id Reference
19717587 D.Kasai, T.Fujinami, T.Abe, K.Mase, Y.Katayama, M.Fukuda, and E.Masai (2009).
Uncovering the protocatechuate 2,3-cleavage pathway genes.
  J Bacteriol, 191, 6758-6768.  
18071645 Y.Huang, K.X.Zhao, X.H.Shen, C.Y.Jiang, and S.J.Liu (2008).
Genetic and biochemical characterization of a 4-hydroxybenzoate hydroxylase from Corynebacterium glutamicum.
  Appl Microbiol Biotechnol, 78, 75-83.  
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
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
11514662 O.Dym, and D.Eisenberg (2001).
Sequence-structure analysis of FAD-containing proteins.
  Protein Sci, 10, 1712-1728.  
9694855 M.H.Eppink, H.A.Schreuder, and W.J.van Berkel (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.
PDB codes: 1bgj 1bgn
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