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Characterization of a novel enzyme system for activation of acetone in sulfate-reducing bacteria

硫酸盐还原菌中丙酮活化的新型酶系统的表征

基本信息

批准号：
391398354
负责人：
Professor Dr. Bernhard Schink
金额：
--
依托单位：
Limnologisches Institut
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2017
资助国家：
德国
起止时间：
2016-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/391398354?language=en
关键词：
Characterization novel enzyme system activation

项目摘要

Whereas acetone-degrading aerobic, phototrophic, and nitrate-reducing bacteria activate their substrate by carboxylation to acetoacetate sulfate-reducing bacteria do not use acetone carboxylase, and acetoacetate is not a free intermediate in the pathway of acetone degradation. Studies with Desulfococcus biacutus in our lab showed that CO is a better co-substrate for acetone degradation than CO2, and that the activation reaction requires coenzyme A, ATP, and thiamine diphosphate as cofactors. According to our present working hypothesis, formyl-CoA is added to the carbonyl carbon of acetone in a thiamine diphosphate-dependent reaction to form 2-hydroxyisobutyryl-CoA which is subsequently isomerized to 3-hydroxybutyryl-CoA and oxidized to acetoacetyl-CoA which, after thiolytic cleavage, is further oxidized through the reversed Wood-Ljungdahl pathway. The planned project will concentrate on purification and characterization of the thiamine diphosphate-dependent 2-hydroxyisobutyryl-CoA synthase and on the formation of the putative co-substrate formyl-CoA. The study can be based on a completely sequenced and annotated genome of D. biacutus, proteomic identification of the enzymes involved in acetone metabolism, broad experience with heterologous expression of enzymes of this bacterium, and excellent chemical and analytical support (HPLC-MS etc.) by partner scientists at Konstanz university.

而丙酮降解好氧，光养，硝酸盐还原菌激活其底物通过羧化乙酰乙酸硫酸盐还原菌不使用丙酮羧化酶，乙酰乙酸不是丙酮降解途径中的游离中间体。我们实验室对双尖脱硫球菌的研究表明，CO是比CO2更好的丙酮降解共底物，并且活化反应需要辅酶A、ATP和二磷酸硫胺素作为辅因子。根据我们目前的工作假设，甲酰基-CoA在硫胺素二磷酸依赖性反应中被添加到丙酮的羰基碳以形成2-羟基异丁酰-CoA，其随后异构化为3-羟基丁酰-CoA并氧化为乙酰乙酰-CoA，其在硫解裂解后通过反向Wood-Ljungdahl途径被进一步氧化。计划中的项目将集中于二磷酸硫胺素依赖的2-羟基异丁酰辅酶A合酶的纯化和表征，以及推定的共底物甲酰辅酶A的形成。该研究可基于已完全测序和注释的D. biacutus，丙酮代谢相关酶的蛋白质组学鉴定，该细菌酶异源表达的广泛经验，以及出色的化学和分析支持（HPLC-MS等）。由康斯坦茨大学的科学家合作完成。