Sau MurA的表征及依布硒啉对Sau MurA和金黄色葡萄球菌的抑制作用  

Study on Characterization of Sau MurA and Inhibition ofEbselen on Sau MurA and Staphylococcus Aureus

作　　者：李明华 马郁芳[2] Li Minghua;Ma Yufang(Jinzhou Medical University,Jinzhou 121000 China;Dalian Medical University,Dalian 116044 China)

机构地区：[1]锦州医科大学,辽宁锦州121000 [2]大连医科大学,辽宁大连116044

出　　处：《锦州医科大学学报》2023年第5期13-19,共7页Journal of Jinzhou Medical University

摘　　要：目的 对金黄色葡萄球菌UDP-N-乙酰葡糖胺烯醇丙酮转移酶(Staphylococcus aureus UDP-N-acetylglucosamine enolpyruvyl transferase,Sau MurA)酶学特性和活性位点进行表征,探究依布硒啉对Sau MurA和金黄色葡萄球菌的抑制作用。方法 构建Sau MurA表达系统,利用亲和层析方法纯化MurA蛋白,通过孔雀石绿检测法对其活性进行鉴定,并对其酶学特性进行分析;利用定点突变方法对MurA的6个特定氨基酸位点进行突变,构建突变蛋白表达体系,确定MurA活性位点。利用依布硒啉对Sau MurA和金黄色葡萄球菌及其成熟生物膜进行处理,探究依布硒啉的抑制作用。结果 确定了Sau MurA具有UDP-N-乙酰葡糖胺烯醇丙酮转移酶活性,在37℃和pH=7.5时表现出最大活性。UDP-N-乙酰葡萄糖胺(UDP-N-Acetyl-glucosamine enolpyruvyl transferase, UNAG)的Km为(0.238±0.043)mM,磷酸烯醇式丙酮酸(phosphoenolpyruvate, PEP)的Km为(0.531±0.046) mM。6个Sau MurA突变蛋白均无活性。依布硒啉对Sau MurA和金黄色葡萄球菌均有抑制作用,半抑制浓度(IC_(50))和最小抑制浓度(MIC)分别是4.9μM,4.28μg/mL。当药物浓度为4×MIC,明显抑制细菌的生长。对于成熟生物膜的抑制作用,16×MIC依布硒啉的抑制率约为80%,相同浓度下的利福平抑制率约为40%。结论 Sau MurA在37℃和pH=7.5时表现出最大活性,具有较低的底物亲和力。确定K22,N23,C119,R124,D308,R400是Sau MurA重要的活性位点。依布硒啉抑制Sau MurA的活性和金黄色葡萄球菌的生长,并有效地去除金黄色葡萄球菌的成熟生物膜。Objective To characterize enzymatic properties and active sites of Staphylococcus aureus UDP-N-acetylglucosamine enolpyruvyl transferase(Sau MurA)and to study the inhibition effect of ebselen on Sau MurA and S.aureus.Methods The expression system of Sau MurA was constructed and MurA protein was purified by affinity chromatography method.The activity of MurA was determined by malachite green method and its enzymatic kinetic properties were analyzed;six amino acids of MurA were mutated by site-directed mutagenesis method and the expression system of mutant proteins was constructed to study active site of MurA.The inhibitory effect of ebselen on Sau MurA and S.aureus as well as mature biofilm of S.aureus was investigated after treatment with ebselen.Results Sau MurA exhibited the activity of UDP-N-Acetylglucosamine enolpyruvyl transferase and showed the maximal activity at 37℃and pH 7.5.The Km for UDP-N-Acetyl-glucosamine enolpyruvyl transferase(UNAG)was 0.238±0.0434 mM and for phosphoenolpyruvate(PEP)was 0.531±0.0461 mM.Six Sau MurA mutant proteins did not show the activity.Ebselen showed the inhibition on the Sau MurA and S.aureus.The half maximal inhibitory concentration(IC 50)and minimum inhibitory concentration(MIC)were 4.9μM and 4.28μg/mL,respectively.When the drug concentration reached 4×MIC,the compound had obviously inhibited the bacteria.For the inhibition of mature biofilm,the inhibition rate of ebselen of 16×MIC was about 80%and the inhibition rate of rifampicin at same concentration was about 40%.Conclusion Sau MurA exhibits maximum activity at 37℃and pH=7.5,with a low substrate affinity.K22,N23,C119,R124,D308 and R400 were identified as important active sites for Sau MurA.Ebselen inhibits the activity of Sau MurA and the growth of S.aureus,and effectively removes mature biofilms of S.aureus.

关 键 词：金黄色葡萄球菌 MurA UDP-N-乙酰氨基葡萄糖烯醇丙酮转移酶 依布硒啉 生物膜 

分 类 号：Q936[生物学—微生物学]