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J. Biol. Chem., Vol. 281, Issue 25, 17044-17053, June 23, 2006

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Kinetic Mechanisms of the Oxygenase from a Two-component Enzyme, p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii^*

Jeerus Sucharitakul¹, Pimchai Chaiyen², Barrie Entsch, and David P. Ballou

From the Department of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Bangkok 10400, Thailand and the Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-06060

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 (C₁) and an oxygenase that uses FMNH^– (C₂). We report detailed transient kinetics studies at 4 °C of the reaction mechanism of C₂.C₂ 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 -FMNH^–2 reacted with oxygen to form C(4a)-hydroperoxy-FMN at 1.1 ± 0.1 x 10⁶ M^–1 s^–1, whereas the C -FMNH^–2 -HPA complex reacted with oxygen to form C(4a)-hydroperoxy-FMN-HPA more slowly (k = 4.8 ± 0.2 x 10⁴ M^–1 s^–1). The kinetic mechanism of C₂ was shown to be a preferential random order type, in which HPA or oxygen can initially bind to the C -FMNH^–2 complex, but the preferred path was oxygen reacting with C -FMNH^–2 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 C₂-C(4a)-hydroxy-FMN-DHPA complex. At high HPA concentrations (>0.5 mM), HPA formed a dead end complex with the C₂-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, C₂ has the unusual ability to use both common flavin cofactors in catalysis.

Received for publication, November 18, 2005 , and in revised form, April 19, 2006.

^* This work was supported by Grant GM64711 (to D. P. B.) from the National Institutes of Health, The Thailand Research Fund Grants RMU4880028 and RTA4780006, and Mahidol University (to P. C.). This study was also supported in part by a Research Team Strengthening Grant from BIOTECH (to Skorn Mongkolsuk). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a scholarship under the Commission on Higher Education Staff Development Project, Chulalongkorn University. Present address: Dept. of Biochemistry, Faculty of Dentistry, Chulalongkorn University.

² To whom correspondence should be addressed: Dept. of Biochemistry and Center for Excellence in Protein Structure & Function, Faculty of Science, Mahidol University, Rama 6 Road, Bangkok 10400, Thailand. Tel.: 662-201-5596; Fax: 662-354-7174; E-mail: scpcy{at}mucc.mahidol.ac.th.

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