Purification and characterization of the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase from Corynebacterium glutamicum

Abstract

Corynebacterium glutamicum ATCC 13032 metabolizes 3-hydroxybenzoate via gentisate. We have now characterized the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase involved in the initial step of 3-hydroxybenzoate catabolism by this strain, a first 3-hydroxybenzoate 6-hydroxylase molecularly and biochemically characterized from a Gram-positive strain. The ncg12923 gene from Corynebacterium glutamicum ATCC 13032 was shown to encode 3-hydroxybenzoate 6-hydroxylase, the enzyme that catalyzes the NADH-dependent conversion of 3-hydroxybenzoate to gentisate. Ncgl2923 was expressed with an N-terminal six-His tag and purified to apparent homogeneity by Ni²(+)-nitrilotriacetic acid affinity chromatography. The purified H₆-Ncgl2923 showed a single band at apparent molecular mass of 49 kDa on a sodium dodecyl sulfate polyacrylamide gel electrophoresis and was found to be most likely a trimer as determined by gel filtration chromatography. It had a specific activity of 6.92 ± 0.39 U mg⁻¹ against 3-hydroxybenzoate and with a K(m) value of 53.4 ± 4.7 μM using NADH as a cofactor. The product formed from the 3-hydroxybenzoate hydroxylation catalyzed by H₆-Ncgl2923 was identified by high-performance liquid chromatography as gentisate, a ring-cleavage substrate in the microbial aromatic degradation. The enzyme exhibited a maximum activity at pH 7.5 in phosphate buffer, and adding flavin adenine dinucleotide to a final concentration of 15 μM would enhance the activity by three-fold. Although this enzyme shares no more than 33% identity with any of reported 3-hydroxybenzoate 6-hydroxylases from Gram-negative bacterial strains, there is little difference in subunit sizes and biochemical characteristics between them.