摘要
细菌采用II型脂肪酸系统合成脂肪酸,其中3-羟脂酰ACP脱水酶催化唯一的脱水反应,是细菌生长的关键酶之一.野油菜黄单胞菌(Xcc)引起几乎所有十字花科植物的黑腐病,在全球范围内造成广泛的经济损失.为研究Xcc中3-羟脂酰ACP脱水酶,本研究利用大肠杆菌3-羟脂酰ACP脱水酶FabZ序列同源比对时,发现其与XC2876(XcfabZ)编码蛋白具有同源性,序列一致性达到46.1%,同时还具有保守的α螺旋结构和活性位点.将XcfabZ异体遗传互补大肠杆菌fabZ(EcfabZ)条件突变株HW7,结果显示添加IPTG能恢复突变株的生长,初步表明XcFabZ具有3-羟脂酰ACP脱水酶活性.而体外活性分析显示,XcFabZ能在脂肪酸合成的起始反应和延伸反应中发挥3-羟脂酰ACP脱水酶活性作用.本研究不能直接获得XcfabZ基因敲除突变株,但将携带EcfabZ或XcfabZ的表达质粒导入后,获得基因替换突变株,证明XcfabZ是必需基因.EcfabZ替换突变株的脂肪酸组成与野生菌有差异,对逆境条件(高盐、低pH、H2O2和SDS)的耐受性显著下降,运动性也显著降低,但XcfabZ替换突变株恢复到野生菌水平,表明XcFabZ与EcFabZ虽然都具有3-羟脂酰ACP脱水酶活性,但在细胞中生理功能可能有一些差别.
In bacteria fatty acids are synthesized by type II fatty acid synthase system,in which 3-hydroxylacyl-ACP dehydratase is one of the key enzymes for bacterial growth.Xanthomonas campestris pv.campestris(Xcc)causes black rot disease to all cruciferous plants,and great economic losses to the world.To study the3-hydroxylacyl-ACP dehydratase in Xcc,XC2876(XcfabZ)was found based on the sequence alignment with E.coli FabZ.Moreover,XcFabZ shows 46.1%amino acid sequence identity with EcFabZ,and contains the conservativeα-helix structure and active residue.XcfabZ was able to genetically complement the EcfabZ knock out mutant E.coli HW7 to restore the growth with inducer IPTG.In vitro assay also identified XcFabZ was able to dehydrated 3-hydroxyacyl-ACP in the initial and elongation reactions.However,XcfabZ in the chromosome could not be deleted directly,indicating XcfabZ is essential for growth.When expression plasmids harboring EcfabZ or XcfabZ was introduced,respectively,XcfabZ deletion mutants were constructed.The EcfabZ replaced mutant showed different fatty acid compositions,much lower tolerance to stressful conditions(high salty,low pH,H2O2 and SDS),and decreased motility compared to the wild-type.While mutant with XcfabZ in the plasmid showed similar phenotype to wild-type.These data demonstrated both XcFabZ and EcFabZ show 3-hydroxylacylACP dehydratase activity,but they may contains different biological function in vivo.
作者
马建荣
陈程
鄢明峰
李先其
张文彬
余永红
MA Jian-Rong;CHEN Cheng;YAN Ming-Feng;LI Xian-Qi;ZHANG Wen-Bin;YU Yong-Hong(Guangdong Food and Drug Vocational College,Guangzhou 510520,China;College of Life Sciences,South China Agricultural University/Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms,Guangzhou 510642,China)
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2021年第6期688-697,共10页
Progress In Biochemistry and Biophysics
基金
广州市科技计划项目(202002030422)
国家自然科学基金(31601601)
广东食品药品职业学院院级课题(2019ZR13,2019ZR17)
广东大学生科技创新培育专项资金(“攀登计划”专项资金)(pdjh2020b1010)
广东省农业生物蛋白质功能与调控重点实验室开放课题(PFRAO201804)资助项目。
关键词
野油菜黄单胞菌
3-羟脂酰ACP脱水酶
脂肪酸合成
Xanthomonas campestris pv.campestris(Xcc)
3-hydroxylacyl-ACP dehydratase
fatty acid synthesis
