Purification and characterization of the ncgl2923 -encoded 3-hydroxybenzoate 6-hydroxylase from Corynebacterium glutamicum
✍ Scribed by Yi-Fan Yang; Jun-Jie Zhang; Song-He Wang; Ning-Yi Zhou
- Publisher
- John Wiley and Sons
- Year
- 2010
- Tongue
- English
- Weight
- 335 KB
- Volume
- 50
- Category
- Article
- ISSN
- 0233-111X
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✦ Synopsis
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 Ni^2+^‐nitrilotriacetic acid affinity chromatography. The purified H~6~‐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 H~6~‐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. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)