COMPARISON OF COBALAMIN-INDEPENDENT AND COBALAMIN-DEPENDENT METHIONINE SYNTHASES FROM ESCHERICHIA-COLI - 2 SOLUTIONS TO THE SAME CHEMICAL PROBLEM
COMPARISON OF COBALAMIN-INDEPENDENT AND COBALAMIN-DEPENDENT METHIONINE SYNTHASES FROM ESCHERICHIA-COLI - 2 SOLUTIONS TO THE SAME CHEMICAL PROBLEM
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DOI：
10.1021/bi00141a013
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发表时间：
1992-07-07
期刊：
影响因子：
2.9
通讯作者：
MATTHEWS, RG
中科院分区：
文献类型：
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作者：
GONZALEZ, JC;BANERJEE, RV;MATTHEWS, RG
在大肠杆菌中，有两种酶催化高半胱氨酸合成甲硫氨酸，使用甲基四氢叶酸作为所需甲基的供体：钴胺素依赖性和钴胺素非依赖性甲硫氨酸合酶。比较这两种酶的机制提供了研究同一化学问题的两种不同解决方案的机会。我们开始了这里描述的研究，以确定是否这两种酶是通过比较推导的氨基酸序列的两种蛋白质的进化相关。我们已经确定了metE基因的核苷酸序列，编码钴胺素非依赖性甲硫氨酸合酶。我们的研究结果揭示了钴胺素依赖和钴胺素独立的蛋白质的推导的氨基酸序列之间的相似性的情况下，并建议，这两个已经出现了趋同进化。我们已经开发了一种快速的一步纯化的重组钴胺素非依赖性甲硫氨酸合酶（MetE），产生均匀的蛋白质在高产量的机制和结构的研究。在这些研究的过程中，我们确定了一个高度反应性的硫醇在MetE是烷基化的氯甲基酮和碘乙酰胺。我们证明，该残基的烷基化，显示为半胱氨酸726，导致活性完全丧失。虽然我们无法推断半胱氨酸726在催化中的作用，在这个时候，这种反应性残基的鉴定表明，这种硫醇功能的可能性，作为一个中间体甲基受体在催化，类似于钴胺素的作用，在反应中催化的钴胺素依赖性酶。
In Escherichia coli, two enzymes catalyze the synthesis of methionine from homocysteine using methyltetrahydrofolate as the donor of the required methyl group: cobalamin-dependent and cobalamin-independent methionine synthases. Comparison of the mechanisms of these two enzymes offers the opportunity to examine two different solutions to the same chemical problem. We initiated the research described here to determine whether the two enzymes were evolutionarily related by comparing the deduced amino acid sequences of the two proteins. We have determined the nucleotide sequence for the metE gene, encoding the cobalamin-independent methionine synthase. Our results reveal an absence of similarity between the deduced amino acid sequences of the cobalamin-dependent and cobalamin-independent proteins and suggest that the two have arisen by convergent evolution. We have developed a rapid one-step purification of the recombinant cobalamin-independent methionine synthase (MetE) that yields homogeneous protein in high yield for mechanistic and structural studies. In the course of these studies, we identified a highly reactive thiol in MetE that is alkylated by chloromethyl ketones and by iodoacetamide. We demonstrated that alkylation of this residue, shown to be cysteine 726, results in complete loss of activity. While we are unable to deduce the role of cysteine 726 in catalysis at this time, the identification of this reactive residue suggests the possibility that this thiol functions as an intermediate methyl acceptor in catalysis, analogous to the role of cobalamin in the reaction catalyzed by the cobalamin-dependent enzyme.