PDBsum entry 2phh

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Oxidoreductase PDB id
Protein chain
391 a.a. *
Waters ×57
* Residue conservation analysis
PDB id:
Name: Oxidoreductase
Title: The coenzyme analogue adenosine 5-diphosphoribose displaces active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation
Structure: P-hydroxybenzoate hydroxylase. Chain: a. Engineered: yes
Source: Pseudomonas fluorescens. Organism_taxid: 294
Biol. unit: Dimer (from PQS)
2.70Å     R-factor:   0.168    
Authors: J.M.Van Derlaan,J.Drenth,W.G.J.Hol
Key ref:
J.M.van der Laan et al. (1989). The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation. Biochemistry, 28, 7199-7205. PubMed id: 2819062 DOI: 10.1021/bi00444a011
19-Jun-89     Release date:   15-Oct-90    
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

 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
Bound ligand (Het Group name = APR)
matches with 71.43% similarity
+ O(2)
= protocatechuate
+ NADP(+)
+ H(2)O
      Cofactor: FAD
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: 10.1021/bi00444a011 Biochemistry 28:7199-7205 (1989)
PubMed id: 2819062  
The coenzyme analogue adenosine 5-diphosphoribose displaces FAD in the active site of p-hydroxybenzoate hydroxylase. An x-ray crystallographic investigation.
J.M.van der Laan, H.A.Schreuder, M.B.Swarte, R.K.Wierenga, K.H.Kalk, W.G.Hol, J.Drenth.
p-Hydroxybenzoate hydroxylase (PHBH) is an NADPH-dependent enzyme. To locate the NADPH binding site, the enzyme was crystallized under anaerobic conditions in the presence of the substrate p-hydroxybenzoate, the coenzyme analogue adenosine 5-diphosphoribose (ADPR), and sodium dithionite. This yielded colorless crystals that were suitable for X-ray analysis. Diffraction data were collected up to 2.7-A resolution. A difference Fourier between data from these colorless crystals and data from yellow crystals of the enzyme-substrate complex showed that in the colorless crystals the flavin ring was absent. The adenosine 5'-diphosphate moiety, which is the common part between FAD and ADPR, was still present. After restrained least-squares refinement of the enzyme-substrate complex with the riboflavin omitted from the model, additional electron density appeared near the pyrophosphate, which indicated the presence of an ADPR molecule in the FAD binding site of PHBH. The complete ADPR molecule was fitted to the electron density, and subsequent least-squares refinement resulted in a final R factor of 16.8%. Replacement of bound FAD by ADPR was confirmed by equilibrium dialysis, where it was shown that ADPR can effectively remove FAD from the enzyme under mild conditions in 0.1 M potassium phosphate buffer, pH 8.0. The empty pocket left by the flavin ring is filled by solvent, leaving the architecture of the active site and the binding of the substrate largely unaffected.

Literature references that cite this PDB file's key reference

  PubMed id Reference
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
  9385648 M.H.Eppink, H.A.Schreuder, and W.J.Van Berkel (1997).
Identification of a novel conserved sequence motif in flavoprotein hydroxylases with a putative dual function in FAD/NAD(P)H binding.
  Protein Sci, 6, 2454-2458.  
8706756 B.Seibold, M.Matthes, M.H.Eppink, F.Lingens, W.J.Van Berkel, and R.Müller (1996).
4-Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3. Purification, characterization, gene cloning, sequence analysis and assignment of structural features determining the coenzyme specificity.
  Eur J Biochem, 239, 469-478.  
7628466 M.H.Eppink, H.A.Schreuder, and W.J.Van Berkel (1995).
Structure and function of mutant Arg44Lys of 4-hydroxybenzoate hydroxylase implications for NADPH binding.
  Eur J Biochem, 231, 157-165.
PDB code: 1bkw
  1729209 B.Averhoff, L.Gregg-Jolly, D.Elsemore, and L.N.Ornston (1992).
Genetic analysis of supraoperonic clustering by use of natural transformation in Acinetobacter calcoaceticus.
  J Bacteriol, 174, 200-204.  
1409567 H.A.Schreuder, J.M.van der Laan, M.B.Swarte, K.H.Kalk, W.G.Hol, and J.Drenth (1992).
Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution.
  Proteins, 14, 178-190.  
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