A novel two-protein component flavoprotein hydroxylase - p-hydroxyphenylacetate hydroxylase from Acinetobacter baumannii

p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) was purified from Acinetoba cter baumannii and shown to be a two-protein component enzyme. The small co mponent (C-1) is the reductase enzyme with a subunit molecular mass of 32 k Da. C-1 alone catalyses HPA-stimulated NADH oxidation without hydroxylation of HPA. C-1 is a flavoprotein with FMN as a native cofactor but can also b ind to FAD. The large component (C-2) is the hydroxylase component that hyd roxylates BPA in the presence of C-1. C-2 is a tetrameric enzyme with a sub unit molecular mass of 50 kDa and apparently contains no redox centre. FMN, FAD, or riboflavin could be used as coenzymes for hydroxylase activity wit h FMN showing the highest activity. Our data demonstrated that C-2, alone, was capable of utilizing reduced FMN to form the product 3,4-dihydroxypheny lacetate. Mixing reduced flavin with C-2 also resulted in the formation of a flavin intermediate that resembled a C(4a)-substituted flavin species ind icating that the reaction mechanism of the enzyme proceeded via C(4a)-subst ituted flavin intermediates. Based on the available evidence, we conclude t hat the reaction mechanism of HPAH from A. baumannii is similar to that of bacterial luciferase. The enzyme uses a luciferase-like mechanism and reduc ed flavin (FMNH2, FADH(2), or reduced riboflavin) to catalyse the hydroxyla tion of aromatic compounds, which are usually catalysed by FAD-associated a romatic hydroxylases.