粪肠球菌(Enterococcus faecalis)β酮脂酰ACP合成酶Ⅱ同源蛋白功能分析

Characterization of β-Ketoacyl-acyl Carrier Protein Synthase Ⅱ Homologues in Enterococcus faecalis

FabB和FabF是大肠杆菌(Escherichia.coli)脂肪酸合成的关键酶.生物信息学分析显示,粪肠球菌基因组中有2个与大肠杆菌fabF同源的基因:fabF1和fabF2,缺少与fabB同源的基因.用粪肠球菌(Enterococcus faecalis)V583总DNA为模板,PCR扩增fabF1和fabF2基因,以pBAD24为载体,构建了重组质粒pHW13(fabF1)和pHW14(fabF2).体内体外研究显示:fabF1基因能互补大肠杆菌fabB突变,FabF1具有β酮脂酰ACP合成酶Ⅰ(FabB)活性;fabF2能互补大肠杆菌fabF突变,FabF2具有β酮脂酰ACP合成酶Ⅱ(FabF)活性.同时发现粪肠球菌FabF2不同于大肠杆菌FabF,它还拥有微弱β酮脂酰ACP合成酶Ⅰ(FabB)活性,可使大肠杆菌fabB突变株产生少量的不饱和脂肪酸.上述结果表明,FabF类酶(FabF like enzyme)同样可以具有β酮脂酰ACP合成酶Ⅰ(FabB)活性.

Abstract FabB （β- ketoacyl-acyl carrier protein synthase Ⅰ ） and FabF（β- ketoacyl-acyl carrier protein synthase Ⅱ ） are two key enzymes of fatty acid biosynthesis in E.coli. The Gram-positive pathogenic bacterium Enterococcus faecalis has a fatty acid composition very similar to that of E.coli. Bioinformatic analysis reveals that though E. faecalis has twofabF homologues, there is no recognizablefabB homologue in the genome of E. faecalis. Two fabF homologues （fabF1 and fabF2） were amplified by using E. faecalis V583 genomitic DNA as template, and two plasmids, pHW13 （fabF1） and pHW14 （fabF2）, were constructed. The results of experiments in vivo and in vitro have shown that fabF1 gene could complement E. coli fabB mutation and FabF1 possessed β- ketoacyl-acyl carrier protein synthase Ⅰ （FabB） activity, while fabF2 gene could complement E. coli fabF mutation and FabF2 had β- ketoacyl-acyl carrier protein synthase Ⅱ （FabF） activity. Meanwhile the data also shown that FabF2 possessed partial function of β-ketoacyl-acyl carrier protein synthase Ⅰ （FabB）, and it could make E. colifabB mutation synthesized low amount of unsaturated fatty acid. From these data it is clear that FabF species enzymes could have activity of β- ketoacyl-acyl carrier protein synthase I （FabB）.