Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

Crystal Structure of 3-Hydroxybenzoate 6-Hydroxylase Uncovers Lipid-assisted Flavoprotein Strategy for Regioselective Aromatic Hydroxylation

“…Consistently, an additional OH group on the lipid-facing edge of the substrate ring makes the binding weaker (3,5-dihydroxybenzoate) or very poor (3,4-dihydroxybenzoate). Only the 2-hydroxyl group, which will bind opposite the lipid wall, can be fully tolerated with 2,3-dihydroxybenzoate, which is essentially as good a substrate as 3-hydroxybenzoate (14). This mode of affording selectivity is nicely illustrated by the comparison of 3HB6H with the substrate-bound structure of 3HB4H (Protein Data Bank code 2DKH).…”

“…Yellow crystals grew in 1 day. A platinum derivative was obtained by soaking the 3HB6H crystals for 16 h in 20 l of reservoir solution containing 0.5 mM (14). One unit of enzyme activity is defined as the amount of enzyme that consumes 1 mol of NADH/min.…”

“…Protein Production-3HB6H from R. jostii RHA1 and 3HB6H variants were expressed in E. coli and purified as C-terminal His 6 -tagged protein as described previously (14).…”

“…Dissociation constants (K d ) of enzyme-substrate complexes were determined from flavin absorption difference spectra essentially as described elsewhere (14).…”

“…Besides 3-hydroxybenzoate, the enzyme converts a limited number of 3-hydroxybenzoate analogs to the corresponding gentisates (14).…”