Mechanism of radical-mediated dehydration reactions in anaerobic bacteria

厌氧菌自由基介导的脱水反应机制

• 批准号: 28545261
• 负责人: Professor Dr. Wolfgang Buckel
• 金额: --
• 依托单位: Laboratory for Microbial Biochemistry
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2006
• 资助国家: 德国
• 起止时间: 2005-12-31 至 2008-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/28545261?language=en
• 关键词: Mechanism; radical; mediated; dehydration; reactions

Usual dehydratases catalyse the elimination of water from 3-hydroxyacyl-CoA. The electron-withdrawing thiol ester acidifies the ¿-hydrogen to pK <21 and enables its removal by basic amino acid side chains. Anaerobic bacteria, however, ferment amino acids via 2- or 4-hydroxyacyl-CoA, whose dehydrations involve the abstraction of a ß-hydrogen, which is ostensibly non-acidic (pK ca. 40). We have proposed mechanisms, in which one-electron transfer generates enoxy radicals with much lower pK-values and in which the elimination of the hydroxy groups involves ketyl radical anions. We want further elucidate these unprecedented mechanisms by site-directed mutagenesis of three participating enzymes: 2-Hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, which is activated by one-electron reduction mediated by ATP, ferredoxin and an archerase or activator. The electron is further transferred to the substrate generating an enoxy radical. In contrast the action of 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum requires transient one electron-oxidation of the substrate to an enoxy radical. In addition we plan to analyse the specificity and stereochemistry of the substrates of these unusual enzymes, which will substantiate conclusions drawn from X-ray crystallography.

乙醛脱氢酶催化从3-羟酰基-CoA中消除水。吸电子硫羟酸酯将π-氢酸化至pK <21，并使其能够通过碱性氨基酸侧链除去。然而，厌氧细菌通过2-或4-羟酰基-CoA发酵氨基酸，其脱水涉及提取表面上非酸性的β-氢（pK ca. 40）。我们已经提出了机制，其中一个电子转移产生具有低得多的pK值的烯氧基自由基，其中羟基基团的消除涉及酮基自由基阴离子。我们希望通过三种参与酶的定点诱变进一步阐明这些前所未有的机制：来自艰难梭菌的2-羟基异己酰-CoA脱氢酶，其通过ATP、铁氧还蛋白和archerase或激活剂介导的单电子还原来激活。电子进一步转移到底物，产生烯氧基自由基。相比之下，来自氨基丁酸梭菌的4-羟基丁酰-CoA脱氢酶的作用需要底物瞬时的一个电子氧化为烯氧基自由基。此外，我们计划分析这些不寻常的酶的底物的特异性和立体化学，这将证实从X射线晶体学得出的结论。