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首页> 外文期刊>Journal of bacteriology >Novel l-Cysteine-Dependent Maleylpyruvate Isomerase in the Gentisate Pathway of Paenibacillus sp. Strain NyZ101
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Novel l-Cysteine-Dependent Maleylpyruvate Isomerase in the Gentisate Pathway of Paenibacillus sp. Strain NyZ101

机译：Paenibacillus sp的Gentis通路中的新型l半胱氨酸依赖的马来酰丙酮酸异构酶。菌株NyZ101

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Glutathione- and mycothiol-dependent maleylpyruvate isomerases are known to be involved, respectively, in gentisate catabolism in Gram-negative and high G+C Gram-positive strains. In the present study, a low-G+C Gram-positive Paenibacillus sp. strain, NyZ101, was isolated and shown to degrade 3-hydroxybenzoate via gentisate. A 6.5-kb fragment containing a conserved region of gentisate 1,2-dioxygenase genes was cloned and sequenced, and four genes (bagKLIX) were shown to encode the enzymes involved in the catabolism to central metabolites of 3-hydroxybenzoate via gentisate. The Bag proteins share moderate identities with the reported enzymes in the 3-hydroxybenzoate catabolism, except BagL that had no obvious homology with any functionally characterized proteins. Recombinant BagL was purified to homogeneity as a His-tagged protein and likely a dimer by gel filtration. BagL was demonstrated to be a novel thiol-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate with l-cysteine, cysteinylglycine, or glutathione, as its cofactor. The Km values of these three thiols for BagL were 15.5, 8.4, and 552 μM, respectively. Since cysteine and coenzyme A were reported to be abundant in low-G+C Gram-positive strains, BagL should utilize l-cysteine as its physiological cofactor in vivo. The addition of Ni2+ increased BagL activity, and site-directed mutagenesis experiments indicated that three conserved histidines in BagL were associated with binding to Ni2+ ion and were necessary for its enzyme activity. BagL is the first characterized l-cysteine-dependent catabolic enzyme in microbial metabolism and is likely a new and distinct member of DinB family, with a four-helix-bundle topology, as deduced by sequence analysis and homology modeling.
机译：已知谷胱甘肽和麦考硫醇依赖性的马来酰丙酮酸异构酶分别与革兰氏阴性和高G + C革兰氏阳性菌株的龙胆酸酯分解代谢有关。在本研究中，低G + C革兰氏阳性Paenibacillus sp。分离出菌株NyZ101，并显示其通过龙胆酸酯降解3-羟基苯甲酸酯。克隆并测序了一个包含龙胆酸酯1,2-二加氧酶基因保守区的6.5kb片段，并显示了四个基因（ bagKLIX ）编码参与分解代谢为3种中心代谢产物的酶-羟基苯甲酸酯通过龙胆酸酯。 Bag蛋白与3-羟基苯甲酸酯分解代谢中所报道的酶具有适度的同一性，除了BagL与任何功能表征的蛋白没有明显同源性。重组的BagL通过His标记的蛋白被纯化至同质，并可能通过凝胶过滤纯化为二聚体。 BagL被证明是一种新型的硫醇依赖性顺丁烯二丙酮酸异构酶，其以L-半胱氨酸，半胱氨酰甘氨酸或谷胱甘肽为辅因子催化顺丁烯二酸丙酮酸异构化为富马酰丙酮酸。这三种硫醇对BagL的 K m 值分别为15.5、8.4和552μM。由于据报道在低G + C革兰氏阳性菌株中半胱氨酸和辅酶A丰富，因此BagL应该利用l-半胱氨酸作为其体内的生理辅助因子。 Ni 2 + 的添加增加了BagL的活性，定点诱变实验表明BagL中的三个保守组氨酸与Ni 2 + 离子的结合有关，这对于它的酶活性。 BagL是微生物代谢中第一个特征化的依赖于半胱氨酸的分解代谢酶，它可能是DinB家族的一个新成员，具有四螺旋束拓扑结构，这是通过序列分析和同源性建模推论得出的。

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