Kinetic mechanisms of the oxygenase from a two-component enzyme, p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii

Abstract

p-Hydroxyphenylacetate hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4-dihydroxyphenylacetate (DHPA). The enzyme system is composed of two proteins: an FMN reductase (C 1) and an oxygenase that uses FMNH - (C 2). We report detailed transient kinetics studies at 4°C of the reaction mechanism of C 2. C 2 binds rapidly and tightly to reduced FMN (K d, 1.2 ± 0.2 μM), but less tightly to oxidized FMN (K d, 250 ± 50 μM). The complex of C 2-FMNH - reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 ± 0.1 × 10 6 M -1 s -1, whereas the C 2-FMNH --HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k = 4.8 ± 0.2 × 10 4 M -1 s -1). The kinetic mechanism of C 2 was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C 2-FMNH - complex, but the preferred path was oxygen reacting with C 2-FMNH - to form the C(4a)-hydroperoxy-FMN intermediate prior to HPA binding. Hydroxylation occurs from the ternary complex with a rate constant of 20 s -1 to form the C 2-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (>0.5 mM),HPA formed a dead end complex with the C 2-C(4a)-hydroxy-FMN intermediate (similar to single component flavoprotein hydroxylases), thus inhibiting the bound flavin from returning to the oxidized form. When FADH - was used, C(4a)-hydroperoxy-FAD, C(4a)-hydroxy-FAD, and product were formed at rates similar to those with FMNH -. Thus, C 2 has the unusual ability to use both common flavin cofactors in catalysis. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.