Reduction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

Biochemistry
Jeerus SucharitakulPimchai Chaiyen

Abstract

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M(-1) s(-1). In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s(-1). At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is ~51 s(-1), whereas without 3-HB, the rate constant is 0.43 s(-1) at a similar NADH concentration. A similar stimulation of flavin reduction wa...Continue Reading

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Dec 24, 2013·Archives of Biochemistry and Biophysics·Mieke M E HuijbersWillem J H van Berkel
Aug 21, 2013·Biochemistry·Austin D Vogt, Enrico Di Cera
Oct 12, 2013·Biophysical Chemistry·Austin D VogtEnrico Di Cera
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Feb 6, 2020·The Journal of Biological Chemistry·Warintra PitsawongAnne-Frances Miller
Jul 4, 2017·Frontiers in Microbiology·Stefania MontersinoWillem J H van Berkel
Dec 15, 2020·Biophysics Reviews·Enrico Di Cera

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