Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In this study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA to butyrate and caproyl-CoA to caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8 times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse β-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp346 and Ala351) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp346 and Ala351 have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production.

中文翻译：

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丁酰/己酰辅酶A：乙酸辅酶A转移酶：参与中链脂肪酸生物合成的关键酶的克隆、表达和表征。

辅酶 A 转移酶 (CoAT) 是参与碳链延伸的重要酶，有助于中链脂肪酸 (MCFA) 的生物合成。例如，丁酰辅酶A：乙酸辅酶A转移酶（BCoAT）负责从丁酰辅酶A合成丁酸的最后一步。然而，关于己酰辅酶A：乙酸辅酶A转移酶（CCoAT）知之甚少，它负责从己酰辅酶A合成己酸的最后一步。本研究通过基因克隆和表达分析鉴定了瘤胃球菌科细菌CPB6和酪丁酸梭菌BEY8的两个CoAT基因。使用丁酰辅酶A或己酰辅酶A作为底物进行酶测定和动力学研究。CPB6-CoAT 可以催化丁酰辅酶 A 转化为丁酸和己酰辅酶 A 转化为己酸，但其以己酰辅酶A为底物的催化效率是丁酰辅酶A的3.8倍。相比之下，BEY8-CoAT 只有 BCoAT 活性，没有 CCoAT 活性。这证明了通过反向 β-氧化途径参与链延长的特定 CCoAT 的存在。比较生物信息学分析表明，CoAT 中存在高度保守的基序 (GGQXDFXXGAXX)，预计该基序是活性中心。CPB6-CoAT 保守基序（Asp346 和 Ala351）的单点突变导致丁酰辅酶 A 和己酰辅酶 A 活性显着降低，表明保守基序是 CPB6-CoAT 的活性中心，而 Asp346 和 Ala351酶活性有显着影响。