Publication: The reductase of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis
Issued Date
2005-08-02
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00062960
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2-s2.0-23044506644
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Mahidol University
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SCOPUS
Bibliographic Citation
Biochemistry. Vol.44, No.30 (2005), 10434-10442
Suggested Citation
Jeerus Sucharitakul, Pimchai Chaiyen, Barrie Entsch, David P. Ballou The reductase of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis. Biochemistry. Vol.44, No.30 (2005), 10434-10442. doi:10.1021/bi050615e Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/16308
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The reductase of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii requires p-hydroxyphenylacetate for effective catalysis
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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 (C2). C1 is a flavoprotein containing FMN, and its function is to provide reduced flavin for C2 to hydroxylate HPA. We have shown here that HPA plays important roles in the reaction of C1. The apoenzyme of C1 binds to oxidized FMN tightly with a Kd of 0.006 μM at 4°C, but with a Kd of 0.038 μM in the presence of HPA. Reduction of C1 by NADH occurs in two phases with rate constants of 11.6 and 3.1 s-1 and Kd values for NADH binding of 2.1 and 1.5 mM, respectively. This result indicates that C1 exists as a mixture of isoforms. However, in the presence of HPA, the reduction of C1 by NADH occurred in a single phase at 300 s-1 with a Kd of 25 μM for NADH binding at 4°C. Formation of the C1-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 C1-HPA complex is -245 mV compared to an E°m of C1 of -236 mV). When the C1-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 C1 with oxygen, the reoxidation reaction is also biphasic, consistent with reduced C1 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. © 2005 American Chemical Society.