PDBsum entry 1cj3

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Oxidoreductase PDB id
Protein chain
392 a.a. *
Waters ×263
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: Mutant tyr38glu of para-hydroxybenzoate hydroxylase
Structure: Protein (p-hydroxybenzoate hydroxylase). Chain: a. Engineered: yes. Mutation: yes. Other_details: complexed with flavin-adenine dinucleotide, hydroxybenzoic acid
Source: Pseudomonas fluorescens. Organism_taxid: 294. Gene: poba. Expressed in: escherichia coli. Expression_system_taxid: 562.
Biol. unit: Dimer (from PDB file)
2.50Å     R-factor:   0.161    
Authors: M.H.M.Eppink,K.M.Overkamp,H.A.Schreuder,W.J.H.Van Berkel
Key ref:
M.H.Eppink et al. (1999). Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase. J Mol Biol, 292, 87-96. PubMed id: 10493859 DOI: 10.1006/jmbi.1999.3015
21-Apr-99     Release date:   30-Apr-99    
Go to PROCHECK summary

Protein chain
Pfam   ArchSchema ?
P00438  (PHHY_PSEFL) -  p-hydroxybenzoate hydroxylase
394 a.a.
392 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: 10.1006/jmbi.1999.3015 J Mol Biol 292:87-96 (1999)
PubMed id: 10493859  
Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.
M.H.Eppink, K.M.Overkamp, H.A.Schreuder, W.J.Van Berkel.
p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase.
  Selected figure(s)  
Figure 2.
Figure 2. Stereoview of the Q34T mutant. A 2 F[o] -F[c]electron density map of mutant Q34T is contoured at 2s with Q34T in open and the wild-type structure in dark bonds.
Figure 3.
Figure 3. Stereoview of the Y38E mutant. A 2 F[o] -F[c]electron density map of mutant Y38E is contoured at 2s with Y38E in dark and the wild-type structure in open bonds.
  The above figures are reprinted by permission from Elsevier: J Mol Biol (1999, 292, 87-96) copyright 1999.  
  Figures were selected by an automated process.  

Literature references that cite this PDB file's key reference

  PubMed id Reference
19911309 C.Y.Kim, C.Webster, J.K.Roberts, J.H.Moon, E.Z.Alipio Lyon, H.Kim, M.Yu, L.W.Hung, and T.C.Terwilliger (2009).
Analysis of nucleoside-binding proteins by ligand-specific elution from dye resin: application to Mycobacterium tuberculosis aldehyde dehydrogenases.
  J Struct Funct Genomics, 10, 291-301.  
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.  
19693930 G.A.Khoury, H.Fazelinia, J.W.Chin, R.J.Pantazes, P.C.Cirino, and C.D.Maranas (2009).
Computational design of Candida boidinii xylose reductase for altered cofactor specificity.
  Protein Sci, 18, 2125-2138.  
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.  
15153101 N.M.Kamerbeek, M.W.Fraaije, and D.B.Janssen (2004).
Identifying determinants of NADPH specificity in Baeyer-Villiger monooxygenases.
  Eur J Biochem, 271, 2107-2116.  
12968028 U.Kirchner, A.H.Westphal, R.Müller, and W.J.van Berkel (2003).
Phenol hydroxylase from Bacillus thermoglucosidasius A7, a two-protein component monooxygenase with a dual role for FAD.
  J Biol Chem, 278, 47545-47553.  
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
11322873 N.M.Kamerbeek, M.J.Moonen, J.G.Van Der Ven, W.J.Van Berkel, M.W.Fraaije, and D.B.Janssen (2001).
4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds.
  Eur J Biochem, 268, 2547-2557.  
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.  
The most recent references are shown first. Citation data come partly from CiteXplore and partly from an automated harvesting procedure. Note that this is likely to be only a partial list as not all journals are covered by either method. However, we are continually building up the citation data so more and more references will be included with time. Where a reference describes a PDB structure, the PDB codes are shown on the right.