The Reductase of p-Hydroxyphenylacetate 3-Hydroxylase from Acinetobacter baumannii Requires p-Hydroxyphenylacetate for Effective Catalysis ² Jeerus Sucharitakul, ‡ Pimchai Chaiyen,* ,‡ Barrie Entsch, § and David P. Ballou § Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol UniVersity, Rama 6 Road, Bangkok, Thailand, and Department of Biological Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109 ReceiVed April 4, 2005; ReVised Manuscript ReceiVed June 13, 2005 ABSTRACT: p-Hydroxyphenylacetate (HPA) hydroxylase (HPAH) from Acinetobacter baumannii catalyzes hydroxylation of HPA to form 3,4-dihydroxyphenylacetate. It is a two-protein system consisting of a smaller reductase component (C 1 ) and a larger oxygenase component (C 2 ). C 1 is a flavoprotein containing FMN, and its function is to provide reduced flavin for C 2 to hydroxylate HPA. We have shown here that HPA plays important roles in the reaction of C 1 . The apoenzyme of C 1 binds to oxidized FMN tightly with a K d of 0.006 μM at 4 °C, but with a K d of 0.038 μM in the presence of HPA. Reduction of C 1 by NADH occurs in two phases with rate constants of 11.6 and 3.1 s -1 and K d values for NADH binding of 2.1 and 1.5 mM, respectively. This result indicates that C 1 exists as a mixture of isoforms. However, in the presence of HPA, the reduction of C 1 by NADH occurred in a single phase at 300 s -1 with a K d of 25 μM for NADH binding at 4 °C. Formation of the C 1 -HPA complex prior to binding of NADH was required for this stimulation. The redox potentials indicate that the rate enhancement is not due to thermodynamics (E° m of the C 1 -HPA complex is -245 mV compared to an E° m of C 1 of -236 mV). When the C 1 -HPA complex was reduced by 4(S)-NADH, the reduction rate was changed from 300 to 30 s -1 , giving a primary isotope effect of 10 and indicating that C 1 is specifically reduced by the pro-(S)- hydride. In the reaction of reduced C 1 with oxygen, the reoxidation reaction is also biphasic, consistent with reduced C 1 being a mixture of fast and slow reacting species. Rate constants for both phases were the same in the absence and presence of HPA, but in the presence of HPA, the equilibrium shifted toward the faster reacting species. p-Hydroxyphenylacetate hydroxylase (HPAH) 1 catalyzes hydroxylation of p-hydroxyphenylacetate (HPA) to form 3,4- dihydroxyphenylacetate (DHPA). This hydroxylation is usu- ally found as the initial step in the aerobic degradation pathway for HPA, one of the major metabolites of lignin (1). In the past decade, it has been revealed that the hydroxylation of HPA in various organisms is carried out by at least three different types of two-protein component enzyme systems (2-4). The first HPAH purified was from Pseudomonas putida and shown to be a two-protein com- ponent enzyme system (2). Studies of P. putida HPAH have shown that FAD is tightly bound to the smaller component and that the larger component appears to be a coupling protein enabling hydroxylation (2, 5). The reaction with NADH and that with oxygen and substrate were reported to occur on the flavoprotein component. The mechanism of P. putida HPAH seems to be similar to the mechanism of single-component aromatic flavoprotein hydroxylases except that two proteins are required (5-7). A different HPAH system was later isolated from Escherichia coli W, and studies have shown that the smaller component of E. coli HPAH (HpaC) is a flavin reductase generating free reduced FAD that is transferred to the larger component (HpaB) to hydroxylate HPA (3, 8). It was shown that several different flavin reductases could replace HpaC in the reaction of E. coli HPAH (8, 9). We have isolated HPAH from Acinetobacter baumannii and showed that the enzyme is quite different from the analogous HPAH enzymes from both P. putida and E. coli (4, 10). The A. baumannii HPAH is a two-protein enzyme system consisting of a smaller reductase component (C 1 ) and a larger oxygenase component (C 2 )(4). The enzyme was ² Financial support was received from The Thailand Research Fund Grants RSA/09/2545 and RTA4780006 and Mahidol University (to P.C.) and NIH Grant GM64711 (to D.P.B.). J.S. is a recipient of a scholarship under the Commission on Higher Education Staff Develop- ment Project, Mahidol University. This study was also partly supported by a Research Team Strengthening Grant from BIOTECH to Skorn Mongkolsuk. * To whom correspondence should be addressed. E-mail: scpcy@ mucc.mahidol.ac.th. Phone: 66-2201-5607. Fax: 66-2354-7174. ‡ Mahidol University. § University of Michigan. 1 Abbreviations: HPA, p-hydroxyphenylacetate; HPAH, p-hydroxy- phenylacetate hydroxylase; C1, reductase component of HPAH from A. baumannii;C2, oxygenase component of HPAH from A. baumannii; HpaC, reductase component of HPAH from E. coli; HpaB, oxygenase component of HPAH from E. coli; NTA, nitrilotriacetate; cB, reductase component of NTA monooxygenase from Aminobacter aminoVorans; PheA, phenol hydroxylase from Bacillus thermoglucosidasius A7; PheA2, reductase component of PheA; C 1 red , reduced form of C1;C1- HPA, complex of C1 and HPA; C1 red -HPA, complex of C1 red and HPA; C1-HPA:NADH, charge-transfer complex of C1-HPA and NADH; C1 red -HPA:NAD + , charge-transfer complex of C1 red -HPA and NAD + ; 4(R)-NADD, deuterated 4(R)-[ 2 H]NADH; 4(S)-NADD, deuterated 4(S)- [ 2 H]NADH; apoC1, apoenzyme of C1; kobs, apparent rate constant; FMN, flavin mononucleotide; FMNH - , reduced flavin mononucleotide. 10434 Biochemistry 2005, 44, 10434-10442 10.1021/bi050615e CCC: $30.25 © 2005 American Chemical Society Published on Web 07/12/2005