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首页> 外文期刊>Biochemistry >Impact of Mutating the Key Residues of a Bifunctional 5,10-Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase from Escherichia coli on Its Activities
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Impact of Mutating the Key Residues of a Bifunctional 5,10-Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase from Escherichia coli on Its Activities

机译：突变的大肠杆菌双功能5,10-亚甲基四氢叶酸脱氢酶-环水解酶关键残基对其活性的影响

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摘要

Methylenetetrahydrofolate dehydrogenase-cyclohydrolase (FolD) catalyzes interconversion of 5,10-methylene-tetrahydrofolate and 10-formyl-tetrahydrofolate in the one-carbon metabolic pathway. In some organisms, the essential requirement of 10-formyl-tetrahydrofolate may also be fulfilled by formyltetrahydrofolate synthetase (Fhs). Recently, we developed an Escherichia coli strain in which the folD gene was deleted in the presence of Clostridium perfringens fhs (E. coli Delta folD/p-fhs) and used it to purify FolD mutants (free from the host-encoded FolD) and determine their biological activities. Mutations in the key residues of E. coli FolD, as identified from three-dimensional structures (D121A, Q98K, K54S, Y50S, and R191E), and a genetic screen (G122D and C58Y) were generated, and the mutant proteins were purified to determine their kinetic constants. Except for the R191E and K54S mutants, others were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. While the R191E mutant showed high cyclohydrolase activity, it retained only a residual dehydrogenase activity. On the other hand, the K54S mutant lacked the cyclohydrolase activity but possessed high dehydrogenase activity. The D121A and G122D (in a loop between two helices) mutants were highly compromised in terms of both dehydrogenase and cyclohydrolase activities. In vivo and in vitro characterization of wild-type and mutant (R191E, G122D, D121A, Q98K, C58Y, K54S, and Y50S) FolD together with three-dimensional modeling has allowed us to develop a better understanding of the mechanism for substrate binding and catalysis by E. coli FolD.
机译：亚甲基四氢叶酸脱氢酶-环水解酶（FolD）催化5，10-亚甲基四氢叶酸和10-甲酰基四氢叶酸在单碳代谢途径中的相互转化。在某些生物中，甲酰基四氢叶酸合成酶（Fhs）也可以满足10-甲酰基四氢叶酸的基本要求。最近，我们开发了一种大肠杆菌菌株，其中在产气荚膜梭菌fhs（E. coli Delta folD / p-fhs）存在的情况下删除了folD基因，并将其用于纯化FolD突变体（不含宿主编码的FolD）和确定其生物活性。从三维结构（D121A，Q98K，K54S，Y50S和R191E）鉴定的大肠杆菌FolD关键残基中的突变，并进行了遗传筛选（G122D和C58Y），并将突变蛋白纯化为确定它们的动力学常数。除了R191E和K54S突变体，其他都在脱氢酶和环水解酶活性方面受到了很大影响。尽管R191E突变体显示出高的环水解酶活性，但它仅保留了残留的脱氢酶活性。另一方面，K54S突变体缺乏环水解酶活性，但是具有高脱氢酶活性。 D121A和G122D（在两个螺旋之间的一个环中）突变体在脱氢酶和环水解酶活性方面都受到严重损害。对野生型和突变体（R191E，G122D，D121A，Q98K，C58Y，K54S和Y50S）的体内和体外表征FolD与三维建模一起使我们对底物结合和分离的机理有了更深入的了解。大肠杆菌FolD催化。

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