PDBsum entry: 1cj3

Oxidoreductase

PDB id: 1cj3

Contents

Protein chain

392 a.a. *

Ligands

FAD

PHB

Waters ×263

* Residue conservation analysis

PDB id:

1cj3

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)

Resolution:

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

Date:

21-Apr-99 Release date: 30-Apr-99

Protein chain

P00438  (PHHY_PSEFL) -  P-hydroxybenzoate hydroxylase

Seq: 394 a.a.

Struc: 392 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.1.14.13.2 - 4-hydroxybenzoate 3-monooxygenase.
• Pathway: Benzoate Metabolism
• Reaction: 4-hydroxybenzoate + NADPH + O₂ = protocatechuate + NADP⁺ + H₂O

4-hydroxybenzoate (Bound ligand (Het Group name = PHB) corresponds exactly) + NADPH + O(2) = protocatechuate + NADP(+) + H(2)O

• Cofactor: FAD

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 

• Biological process: metabolic process (4 terms)
• Biochemical function: oxidoreductase activity (4 terms)

reference

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.

ABSTRACT

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.