1CJ2

MUTANT GLN34ARG OF PARA-HYDROXYBENZOATE HYDROXYLASE

Summary for 1CJ2

• Entry DOI: 10.2210/pdb1cj2/pdb
• Descriptor: PROTEIN (P-HYDROXYBENZOATE HYDROXYLASE), FLAVIN-ADENINE DINUCLEOTIDE, P-HYDROXYBENZOIC ACID, ... (4 entities in total)
• Functional Keywords: oxidoreductase, hydroxybenzoate
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 1
• Total formula weight: 44945.86

Authors

Eppink, M.H.M.,Overkamp, K.M.,Schreuder, H.A.,Van Berkel, W.J.H. (deposition date: 1999-04-21, release date: 1999-04-30, Last modification date: 2023-08-09)

Primary citation

Eppink, M.H.,Overkamp, K.M.,Schreuder, H.A.,Van Berkel, W.J.
Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.
J.Mol.Biol., 292:87-96, 1999

PubMed 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.

PubMed: 10493859
DOI: 10.1006/jmbi.1999.3015

Experimental method

X-RAY DIFFRACTION (2.8 Å)