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	Purification and characterization of the ncgl2923-encoded 3-hydroxybenzoate 6-hydroxylase from corynebacterium glutamicum
	Yang, Yi-Fan; Zhang, Jun-Jie; Wang, Song-He; Zhou, Ning-Yi
刊名	Journal of basic microbiology
	2010-12-01
卷号	50期号:6页码:599-604
关键词	3-hydroxybenzoate 6-hydroxylase Corynebacterium glutamicum Characterization Purification
ISSN号	0233-111X
DOI	10.1002/jobm.201000053
通讯作者	Zhou, ning-yi(n.zhou@pentium.whiov.ac.cn)
英文摘要	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 mu m 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 mu m 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.
WOS关键词	METAL CHELATE ADSORBENT ; GENTISATE PATHWAY ; TAGGED PROTEINS ; BINDING ; IDENTIFICATION ; MONOOXYGENASE
WOS研究方向	Microbiology
WOS类目	Microbiology
语种	英语
出版者	WILEY-V C H VERLAG GMBH
WOS记录号	WOS:000285427400011
内容类型	期刊论文
URI标识	http://www.corc.org.cn/handle/1471x/2375801
专题	武汉病毒研究所
通讯作者	Zhou, Ning-Yi
作者单位	Chinese Acad Sci, Wuhan Inst Virol, Key Lab Agr & Environm Microbiol, Wuhan 430071, Peoples R China
推荐引用方式 GB/T 7714	Yang, Yi-Fan,Zhang, Jun-Jie,Wang, Song-He,et al. Purification and characterization of the ncgl2923-encoded 3-hydroxybenzoate 6-hydroxylase from corynebacterium glutamicum[J]. Journal of basic microbiology,2010,50(6):599-604.
APA	Yang, Yi-Fan,Zhang, Jun-Jie,Wang, Song-He,&Zhou, Ning-Yi.(2010).Purification and characterization of the ncgl2923-encoded 3-hydroxybenzoate 6-hydroxylase from corynebacterium glutamicum.Journal of basic microbiology,50(6),599-604.
MLA	Yang, Yi-Fan,et al."Purification and characterization of the ncgl2923-encoded 3-hydroxybenzoate 6-hydroxylase from corynebacterium glutamicum".Journal of basic microbiology 50.6(2010):599-604.

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