Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD( P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a con served FAD-binding domain but lack a recognizable fold for binding the pyri dine nucleotide. We have switched the coenzyme specificity of strictly NADP H-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-4 0) of the FAD-binding domain. Non-conservative selective replacements of Ar g33 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 ex tending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADP H. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of boun d NADPH, and that introduction of an acidic group at position 38 potentiall y enables the recognition of the 2'-hydroxy group of NADH. This is the firs t report on the coenzyme reversion of a flavoprotein aromatic hydroxylase. (C) 1999 Academic Press.