Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 498864
Title Tyr217 and His213 are important for substrate binding and hydroxylation of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1
Author(s) Sucharitakul, Jeerus; Medhanavyn, Dheeradhach; Pakotiprapha, Danaya; Berkel, Willem J.H. Van; Chaiyen, Pimchai
Source FEBS Journal 283 (2016)5. - ISSN 1742-464X - p. 860 - 881.
DOI http://dx.doi.org/10.1111/febs.13636
Department(s) Biochemistry
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2016
Keyword(s) 3-hydroxybenzoate 6-hydroxylase - FAD - hydroxylation - monooxygenase - pre-steady-state kinetics
Abstract

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3HB. Y217A and Y217S did not show any perturbation of flavin absorption upon addition of 3HB, whereas Y217F has a Kd value for 3HB binding of 7.5 mm, which is ~ 50-fold larger than that found for wild-type enzyme. The results clearly indicate that Tyr217 is necessary for substrate binding. All His213 variants can bind to 3HB with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. H213S, H213D and H213E produce 2,5-DHB with yields of 28 ± 5%, 52 ± 7% and 92 ± 6%, respectively, whereas H213A cannot catalyze hydroxylation. The results indicate that the interaction between the hydroxyl group of 3HB and residue 213 is important for substrate hydroxylation. Interestingly, the hydroxylation rate constant of H213E (35 s-1) is similar to that of wild-type enzyme (36 s-1) and this variant has an efficiency of hydroxylation (92 ± 6%) similar to the wild-type enzyme (86 ± 2%). Difference spectra of enzyme-bound substrate suggest that 3HB binds to H213E in the phenolic form. The results indicate that His213 and Glu213 in H213E may act as a catalytic base to initiate the substrate deprotonation and facilitate the electrophilic aromatic substitution of 3HB.

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