野油菜黄单胞菌中烯脂酰ACP还原酶的功能鉴定

烯脂酰ACP还原酶是细菌脂肪酸合成的关键酶之一.本研究通过生物信息学分析发现,野油菜黄单胞菌Xanthomonas campestris(Xcc)8004基因组中XC_0119(Xccfab V)注释为反-2-烯脂酰Co A还原酶基因.但其编码产物与铜绿假单胞菌的烯脂酰ACP还原酶Fab V具有较高的同源性,并含有相同的催化活性中心Tyr-(Xaa)8-Lys序列.用携带Xccfab V的质粒载体互补大肠杆菌fab I温度敏感突变株JP1111,转化子能在42℃生长,表明Xccfab V能遗传互补大肠杆菌fab I突变.体外重建脂肪酸合成反应表明,Xcc Fab V能催化不同链长的烯脂酰ACP还原为脂酰ACP,且催化活性不受三氯森抑制.遗传学研究表明,Xccfab V是必需基因,不能获得Xccfab V基因敲除突变株.将携带大肠杆菌fab I的外源质粒导入野生菌后,可敲除染色体上的fab V基因,获得的替换突变株生长特性和脂肪酸组成未发生显著变化,但替换突变株对三氯森敏感.上述结果证实,野油菜黄单胞菌fab V是必需基因,编码烯脂酰ACP还原酶,参与脂肪酸从头合成反应,且Fab V是Xcc对三氯森耐受的根本原因.

Identification and Function Research of The Enoyl-ACP Reductase in Xanthomonas campestris

Enoyl-ACP reductase (ENR) is one of the key enzymes in bacterial fatty acids biosynthesis. Through sequence alignment, gene XC_0119, annotated as trans-2-enoyl CoA reductase, was found in the genome of Xanthomonas campestris pv. campestris (Xcc) 8004. However, the protein encoded by XC_0119 shows 59% identity with fabV, the ENR from Pseudomonas aeruginosa, and contains the same catalytic triad Tyr-(Xaa)₈-Lys. Expression of XccfabV restored the growth of the E. coli fabI temperature sensitive mutant JP1111 under non-permissive condition. In vitro assay identified that XccFabV catalysed enoyl-ACP with viarable chain lengths to acy-ACP, and the activity was not inhibited by triclosan. Furthermore, genetic research proved that XccfabV is an essential gene, and none of XccfabV deletion mutants was obtained. However, XccfabV in the chromosome could be deleted when plasmid expressing E. coli fabI was introduced into Xcc8004. And the fabI replaced mutant showed similar growth characteristic and fatty acid compositions with wild type, but changed to be sensitive to triclosan. These results domonstrated XccfabV is essential for growth, encodes ENR involved in fatty acid de novo biosynthesis, and FabV confers triclosan resistance on Xcc.