Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1824, Issue 3, Pages 433-442Publisher
ELSEVIER
DOI: 10.1016/j.bbapap.2011.12.003
Keywords
Fingerprint; Flavoprotein; Functional annotation; 3-hydroxybenzoate 6-hydroxylase; Monooxygenase; Rhodococcus jostii RHAl
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Funding
- Integrated Biosynthesis Organic Synthesis (IBOS) of the Netherlands Organization for Scientific Research (NWO)
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The genome of Rhodococcus jostii RHAl contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present a multiple sequence alignment and phylogenetic analysis of putative R. jostii RHAl flavoprotein hydroxylases. Out of 18 candidate sequences, three hydroxylases are absent in other available Rhodococcus genomes. In addition, we report the biochemical characterization of 3-hydroxybenzoate 6-hydroxylase (3HB6H), a gentisate-producing enzyme originally mis-annotated as salicylate hydroxylase. R. jostii RHA1 3HB6H expressed in Escherichia coli is a homodimer with each 47 kDa subunit containing a non-covalently bound FAD cofactor. The enzyme has a pH optimum around pH 8.3 and prefers NADH as external electron donor. 3HB6H is active with a series of 3-hydroxybenzoate analogues, bearing substituents in ortho- or meta-position of the aromatic ring. Gentisate, the physiological product, is a non-substrate effector of 3HB6H. This compound is not hydroxylated but strongly stimulates the NADH oxidase activity of the enzyme. (c) 2011 Elsevier B.V. All rights reserved.
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