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

尽管降解丙酮的好氧、光养和硝酸盐还原细菌通过羧化成乙酰乙酸硫酸盐来激活它们的底物，但还原细菌不使用丙酮羧化酶，并且乙酰乙酸在丙酮降解途径中不是一个自由的中间体。我们实验室对双角脱硫球菌的研究表明，CO是比CO2更好的丙酮降解共底物，其活化反应需要辅酶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，甲酰基辅酶a在硫胺素二磷酸依赖反应中加入到丙酮的羰基碳上形成2-羟基异丁基辅酶a，该辅酶a随后异构化为3-羟基丁基辅酶a，并被氧化为乙酰乙酰辅酶a，乙酰乙酰辅酶a在硫解裂解后通过反向Wood-Ljungdahl途径进一步被氧化。计划中的项目将集中于硫胺素二磷酸依赖的2-羟基异丁基辅酶a合成酶的纯化和表征，以及推定的共底物甲酰基辅酶a的形成。这项研究可以基于D. biacutus基因组的完整测序和注释，参与丙酮代谢的酶的蛋白质组学鉴定，该细菌酶的异源表达的广泛经验，以及康斯坦茨大学的合作科学家提供的出色的化学和分析支持（HPLC-MS等）。

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.