Conversion of Organofluorine Compounds with Microbial Enzymes : Mechanistic Analysis of the Enzyme Reactions and Their Application to Production of Useful Compounds and Bioremediation of Environments

1.氟乙酸酯脱卤酶催化氟乙酸酯的水解脱氟生成乙醇酸。酶的D104亲核攻击底物的α-碳原子，释放氟离子，形成酯中间体，底物衍生的部分与酶共价结合。中间体被H271激活的水分子水解，释放乙醇酸并再生D104。对野生型酶、D104N突变酶与氟乙酸酯络合(Michaelis络合物)、11271A型突变酶与氯乙酸酯(酯中间体)络合的X-射线晶体分析支持该反应机理。底物的羧酸基与H149、W150、Y212、R105和R108结合，氟原子与R108结合。我们发现，W150F突变体酶不作用于氟乙酸酯，而突变体对氯乙酸酯的活性与野生型酶相似，表明W150是脱氟所必需的。以1，1，1-三氯-2，2，2-三氟乙烷(氟利昂113a)为电子受体，富集培养得到一株硫代螺杆菌属厌氧菌。通过反式氯乙烯作为电子受体的浓缩培养，我们获得了一株厌氧细菌，其16S rRNA序列与未培养细菌的16S rRNA序列具有98%的同源性。从伯克霍尔德氏菌出发，构建了一株产2-氯丙烯酸酯还原酶的重组大肠杆菌。WS和葡萄糖脱氢酶，作为NADPH再生酶。利用重组大肠杆菌细胞不对称还原2-氯丙烯酸制得除草剂前驱体(S)-2-氯丙酸酯。该方法在产品收率方面优于传统方法(光学拆分法)。

1. Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. D104 of the enzyme nucleophilically attacks the α-carbon atom of the substrate to release the fluoride ion and form an ester intermediate, in which the substrate-derived moiety is covalently bound to the enzyme. The intermediate is hydrolyzed by a water molecule activated by H271 to release glycolate and regenerate D104. X-ray crystallographic analysis of the wild-type enzyme, the D104N mutant enzyme complexed with fluoroacetate (Michaelis complex), and the 11271A mutant enzyme complexed with chloroacetate (ester intermediate) supported the proposed reaction mechanism. The carboxylate group of the substrate was bound to H149, W150, Y212, R105, and R108, and the fluorine atom was bound to R108. We found that the W150F mutant enzyme does not act on fluoroacetate, whereas the activity of the mutant toward chloroacetate was similar to that of the wild-type enzyme, indicating that W150 is specifically required for the defluorination.2. We obtained an anaerobic bacterium belonging to the genus Sulfurospirillum by enrichment culture with 1,1,1-trichloro-2,2,2-trifluoroethane (Freon 113a) as an electron acceptor. By enrichment culture with retrachlorcethene as an electron acceptor, we obtained an anaerobic bacterium whose 16S rRNA sequence shows 98% identity with that of an uncultured bacterium.3. We constructed a recombinant Escherichia coli strain producing 2-chloroacrylate reductase from Burkholderia sp. WS and glucose dehydrogenase, which serves as an NADPH regeneration enzyme. (S)-2-Chloropropionate, a precursor for the synthesis of herbicides, was produced by asymmetric reduction of 2-chloroacrylate by using the recombinant E. coli cells. This method was superior to the conventional method (optical resolution) in the yield of the product.