五个3-酮脂酰ACP合成酶同源蛋白的功能鉴定  

Identification and Function Reasearch of Five3-Ketoacyl-ACP Synthase Homologues

作　　者：马金成[1] 邓丽婷[1] 童文华[1] 朱磊[1] 王海洪[1] 

机构地区：[1]华南农业大学生命科学学院,广东省农业生物蛋白质功能与调控重点实验室,广州510642

出　　处：《生物化学与生物物理进展》2014年第9期887-895,共9页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金(31200028);高等学校博士学科点专项科研基金(20104404110005);广东高校优秀青年创新人才培养计划(LYM10038)资助项目~~

摘　　要：大肠杆菌的FabB和FabF均具有长链3-酮基脂酰ACP合成酶活性.除参与长链饱和脂酰链的延伸外,FabB还是合成不饱和脂肪酸的关键酶之一,参与不饱和脂酰ACP的从头合成,最终生成顺-9-十六烯脂酰ACP.而FabF只能将顺-9-十六烯脂酰ACP延伸为顺-11-十八烯脂酰ACP,不参与不饱和脂酰ACP的从头合成.有研究表明,粪肠球菌、乳酸乳球菌、丙酮丁醇梭菌和茄科雷尔氏菌等细菌的FabF同源蛋白,具有类似大肠杆菌FabB和FabF的双功能.为证实该现象是否普遍存在,本研究选取了枯草芽孢杆菌BsfabF、中华苜蓿根瘤菌SmfabF、霍乱弧菌VcfabF、铜绿假单胞菌PafabF1和PafabF2 5个同源基因进行功能鉴定,体外酶学分析表明,5个FabF同源蛋白均具有长链3-酮基脂酰ACP合成酶活性,异体互补大肠杆菌CL28的脂肪酸组分分析显示,SmfabF、VcfabF、PafabF1和PafabF2具有3-酮脂酰ACP合成酶Ⅱ(FabF)活性,遗传互补大肠杆菌温度敏感突变株CY242和CY244的研究显示,仅有PafabF2编码的蛋白拥有3-酮脂酰ACP合成酶Ⅰ(FabB)活性,能互补大肠杆菌fabB的突变.这表明不是所有的FabF同源蛋白均具有3-酮脂酰ACP合成酶Ⅰ和Ⅱ的双重活性.FabB and FabF are two key long-chain 3-ketoacyl-ACP synthase in Escherichia coli. In addition to participating synthesis of long acyl chains, FabB is a key enzyme responsible for a condensation reaction in de novo unsaturated fatty acid synthesis and to form palmitoleoyl-ACP. While the FabF was found to be required for the elongation of cis-9-hexadecenoyl-ACP to c/s-11-octadecenoyl-ACP and not involved in de novo unsaturated fatty acid synthesis. It has been reported previously that FabF homologues were found to have both KAS Ⅰ and Ⅱ activity just like FabB and FabF of E. coli in Enterococcus faecalis, Lactococcus lactis, Clostridiurn acetobutylicium and Ralstonia solanacearum. To test if this phenomenon is prevalent, we have carded out functional identification of fivefabF homologous genes, Bacillus subtilis Bsfab F, Sinorhizobium meliloti Smfab F, Vibrio cholera VcfabF, Pseudomonas acruginosa PafabF1 and PafabF2. Our data revealed that five FabF homologues all have the long-chain 3-ketoacyl-ACP synthase activities in vitro. Analysis of phospholipid compositions show that SmfabF, VcfabF, PafabF1 and PafabF2 possessed 3-ketoacyl-ACP synthase 1I （FabF） activity when complemented the E. coli fabF mutation CL28. The results of genetic complementation and thin-layer chromatographic analysis show that only PafabF2 gene could complement E. coli fabB mutation and PaFabF2 possessed partial function of 3-ketoacyl-ACP synthase I （FabB）. These results demonstrated that not all FabF homologues have dual activity of KAS Ⅰ and KAS Ⅱ.

关 键 词：细菌脂肪酸合成 3-酮基脂酰ACP合成酶Ⅰ 3-酮脂酰ACP合成酶Ⅱ 遗传互补 

分 类 号：Q93[生物学—微生物学]