大肠杆菌3-酮基脂酰ACP还原酶110位天冬酰胺突变后的结构与功能

Structure and Function of N110Q and N110L Mutants of 3-Ketoacyl-ACP Reductase in Escherichia coli

3-酮基脂酰ACP还原酶催化3-酮基脂酰ACP还原为3-羟基脂酰ACP,是细菌脂肪酸合成反应的关键酶之一.为了明确该酶中110位的保守天冬酰胺残基在酶催化活性和酶结构中的作用,本研究采用基因定点突变和蛋白质表达纯化技术,获得了大肠杆菌3-酮基脂酰ACP还原酶FabG的两个突变蛋白:FabG N110Q和FabG N110L.圆二色谱结果显示,天冬酰胺残基的突变改变了FabG的空间结构,使突变蛋白的α螺旋结构明显增加.以3-酮脂酰ACP为底物的酶活性测定表明,突变蛋白的酶活性均有下降,但残存的酶活性达到了FabG的75%以上.突变蛋白FabG N110Q和FabG N110L具有3-酮基脂酰ACP还原酶的活性,能在体外重建细菌脂肪酸合成反应.对fabG温度敏感突变株的遗传互补分析表明,FabG蛋白110位天冬酰胺突变为谷氨酰胺或亮氨酸后,在一定的条件下仍能互补大肠杆菌的生长.本研究结果提示,FabG 110位的天冬酰胺残基不是参与3-酮基脂酰ACP还原酶催化反应的必需氨基酸,它只是作为结构氨基酸,在维持FabG的空间结构的稳定性方面起作用.

Escherichia coli fabG encoded 3-ketoacyl-acyl carrier protein（ACP） reductase is responsible for the first reductive step of the fatty acid synthetic cycle in the type Ⅱ fatty acid synthase system.To investigate the role of conserved FabG Asn110 residue in enzyme catalysis activity and essential structure,two E.coli FabG mutant proteins（FabG N110Q and FabG N110L） were obtained using gene site-directed mutagenesis and nickel chelate chromatography protein purification.The 3-ketodecanoyl-ACP reductase activities of mutant proteins were analyzed using several substrates.The functions of mutant proteins in fatty acid synthesis were determined by reconstituting fatty acid synthetic reaction in vitro.The complementation E.coli fabG（ts） muntant CL104 with fabG mutation gene was analyzed.The results demonstrated that Asn-110 is an essential structural residue and does not directly participate in catalysis reaction.