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

而降解丙酮的好氧细菌、光营养细菌和硝酸盐还原细菌通过羧化为乙酰乙酸酯来激活底物，而硫酸盐还原细菌不使用丙酮羧基酶，乙酰乙酸酯也不是丙酮降解途径中的游离中间体。本实验室对双尖球藻的研究表明，CO是一种比CO2更好的丙酮降解共底物，活化反应需要辅酶A、ATP和硫胺素二磷酸作为辅助因子。根据我们目前的工作假设，在硫胺素二磷酸依赖的反应中，甲酰辅酶A加到丙酮的羰基碳上，生成2-羟基异丁酰辅酶A，后者随后异构化为3-羟基丁酰辅酶A，并氧化为乙酰乙酰辅酶A，硫解裂解后，进一步通过反向Wood-Ljugdahl途径氧化。计划中的项目将集中于依赖于硫胺素二磷酸的2-羟基异丁酰辅酶A合成酶的纯化和表征，以及可能的共底物甲酰-辅酶A的形成。这项研究的基础可以是双尖盘藻完整的测序和注释基因组，参与丙酮代谢的酶的蛋白质组学鉴定，这种细菌的酶的异源表达的丰富经验，以及出色的化学和分析支持(高效液相-质谱分析等)。由康斯坦茨大学的科学家合作完成。

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.