The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum
The gene ncgl2918 encodes a novel maleylpyruvate isomerase that needs mycothiol as cofactor and links mycothiol biosynthesis and gentisate assimilation in Corynebacterium glutamicum
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DOI：
10.1074/jbc.m513192200
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发表时间：
2006-04-21
影响因子：
4.8
通讯作者：
Liu, SJ
中科院分区：
文献类型：
--
作者：
Feng, J;Che, YS;Liu, SJ
谷氨酸棒杆菌基因组的数据挖掘发现了4个与结核分枝杆菌和污垢分枝杆菌的msha、mshB、mhc和mshD基因相似的基因，它们参与了霉硫醇的生物合成。在本研究中，对这些基因进行了个体缺失。突变体mshc(-)和mshD(-)失去了产生真菌硫醇的能力，但突变体mshB(-)与野生型一样产生真菌硫醇。突变株mshc(-)和mshD(-)的表型与野生型相同，但mshc(-)和mshD(-)不能在龙胆酸或3-羟基苯甲酸为碳源的矿物培养基中生长。C.谷氨酸通过不依赖谷胱甘肽的龙胆酸途径吸收龙胆酸和3-羟基苯甲酸。在本研究中发现，在不依赖谷胱甘肽的龙胆酸途径中催化顺丁烯二酰丙酮酸转化为延胡索丙酮酸的顺丁烯二酰丙酮酸异构酶需要麦硫醇作为辅因子。该麦硫醇依赖的马来酸丙酮酸异构酶基因(Ncgl2918)被克隆、活性表达并从大肠杆菌中纯化。纯化的麦硫醇依赖异构酶是一个34 kDa的单体。测得马来酸丙酮酸的表观K-m和V-max分别为148.4+/-11.9mU M和1520+/-57.4mU/min/mg(霉硫醇浓度为2.5mU/M)。先前的研究表明，霉菌硫醇在链霉菌和分枝杆菌中对氧化化学物质和抗生素的解毒作用。据我们所知，这是首次证明真菌硫醇对以龙胆酸或3-羟基苯甲酸为碳源的谷氨酸菌的生长是必需的，并首次表征了依赖真菌硫醇的马来基丙酮酸异构酶。
Data mining of the Corynebacterium glutamicum genome identified 4 genes analogous to the mshA, mshB, mshC, and mshD genes that are involved in biosynthesis of mycothiol in Mycobacterium tuberculosis and Mycobacterium smegmatis. Individual deletion of these genes was carried out in this study. Mutants mshC(-) and mshD(-) lost the ability to produce mycothiol, but mutant mshB(-) produced mycothiol as the wild type did. The phenotypes of mutants mshC(-) and mshD(-) were the same as the wild type when grown in LB or BHIS media, but mutants mshC(-) and mshD(-) were not able to grow in mineral medium with gentisate or 3-hydroxybenzoate as carbon sources. C. glutamicum assimilated gentisate and 3-hydroxybenzoate via a glutathione-independent gentisate pathway. In this study it was found that the maleylpyruvate isomerase, which catalyzes the conversion of maleylpyruvate into fumarylpyruvate in the glutathione-independent gentisate pathway, needed mycothiol as a cofactor. This mycothiol-dependent maleylpyruvate isomerase gene (ncgl2918) was cloned, actively expressed, and purified from Escherichia coli. The purified mycothiol-dependent isomerase is a monomer of 34 kDa. The apparent K-m and V-max values for maleylpyruvate were determined to be 148.4 +/- 11.9 mu M and 1520 +/- 57.4 mu mol/min/mg, respectively (mycothiol concentration, 2.5 mu M). Previous studies had shown that mycothiol played roles in detoxification of oxidative chemicals and antibiotics in streptomycetes and mycobacteria. To our knowledge, this is the first demonstration that mycothiol is essential for growth of C. glutamicum with gentisate or 3-hydroxybenzoate as carbon sources and the first characterization of a mycothiol-dependent maleylpyruvate isomerase.