Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.
The genome of Rhodococcus jostii RHA1 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 RHA1 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 47kDa 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.
Wageningen University, Wageningen, the Netherlands.
Stefania
S
Montersino
Willem J H
WJ
van Berkel
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D016680:Genome, Bacterial; D005841:Gentisates / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010802:Phylogeny; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism; D012240:Rhodococcus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D013379:Substrate Specificity
D001426:Bacterial Proteins; D005841:Gentisates; D062385:Hydroxybenzoates; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; C032948:3-hydroxybenzoic acid; D006899:Mixed Function Oxygenases; C023315:3-hydroxybenzoate-6-hydroxylase