LYS42 AND SER42 VARIANTS OF P-HYDROXYBENZOATE HYDROXYLASE FROM PSEUDOMONAS-FLUORESCENS REVEAL THAT ARG42 IS ESSENTIAL FOR NADPH BINDING

The conserved Arg42 of the flavoprotein p-hydrosybenzoate hydroxylase is located at the entrance of the active site in a loop between helix H2 and sheet E1 of the FAD-binding domain. Replacement of Arg42 by Lys or Ser decreases the turnover rate of p-hydroxybenzoate hydroxylase f rom Pseudomonas fluorescens by more than two orders of magnitude. Rapi d reaction kinetics show that the low activity of the Arg42 variants r esults from impaired binding of NADPH. In contrast to an earlier concl usion drawn for p-hydroxybrnzoate hydroxylase from Acinetobacter-calco aceticus, substitution of Arg42 with Ser42 in the enzyme from P. fluor escens hardly disturbs the binding of FAD. Crystals of [Lys42]p-hydrox ybenzoate hydroxylase complexed with 4-hydroxybenzoate diffract to 0.2 2-nm resolution. The structure of the Lys42 variant is virtually indis tinguishable from the native enzyme with the flavin ring occupying the interior position within the active site. Lys42 in the mutant structu re interacts indirectly via a solvent molecule with the 3-OH of the ad enosine ribose moiety of FAD. Substrate perturbation difference spectr a suggest that the Arg42 replacements influence the solvent accessibil ity of the flavin ring in the oxidized enzyme. In spite of this, the A rg42 variants fully couple enzyme reduction to substrate hydroxylation . Sequence-comparison studies suggest that Arg42 is involved in bindin g of the 2'-phosphoadenosine moiety of NADPH.