介导含硫多肽类抗生素耐药的23S rRNA甲基转移酶NHR的生化功能  

The mechanism of thiopeptin antibiotics resistance-research on the biochemical properties of 23S rRNA methyltransferase

作　　者：沈妍[1] 王哲[1] 陈东戎[1] 

机构地区：[1]复旦大学生物医学研究院药物与结构生物学教研室,上海200032

出　　处：《复旦学报（医学版）》2011年第3期189-193,215,共6页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金青年基金项目(SN30900018)

摘　　要：目的研究23S rRNA甲基转移酶-硫链丝菌肽抗药性RNA甲基转移酶(thiostrepton-resistance-RNA methyltransferase,TSR)和23S rRNA甲基转移酶-那西肽抗药性RNA甲基转移酶(nosiheptide-resistance-RNA methyltransferase,NHR)的生化功能,探讨细菌对含硫多肽类抗生素产生耐药性的分子机制。方法构建克隆,体外表达并纯化TSR和NHR蛋白;利用T7聚合酶体外转录系统体外转录生成23S rRNA底物片段;构建RNA甲基转移酶活性的体外检测体系,在14C-SAM存在的情况下,利用液体闪烁计数仪测定相应底物RNA被甲基化的程度;通过Gel shift方法观察RNA底物与TSR或NHR蛋白的体外相互结合作用。结果得到体外表达的纯化TSR、NHR蛋白及RNA底物;TSR蛋白和NHR蛋白均能将RNA底物甲基化,且NHR甲基转移酶的活性更强;TSR和NHR蛋白与RNA底物均有结合,结合强度均随蛋白浓度增加而增强。结论 TSR和NHR蛋白都能将23S rRNA的特异位点1067A甲基化,并能诱导细菌的抗生素耐药性。Objective To study the biochemical properties of 23S rRNA methyltransferase in vitro,and to discuss the mechanism of thiopeptin antibiotics resistance. Methods The clones were constructed to expresse and purify thiostrepton-resistance-RNA methyltransferase（TSR） and nosiheptide-resistance-RNA methyltransferase（NHR）.RNA substrate was transcripted via T7 polymerase system.Detect system of RNA methyltransferase activity was set up to measure the methylation extent of RNA substrate via liquid scintillation counter with existence of ^14C-SAM.The binding between RNA substrate and TSR or NHR protein was observed via Gel shift. Results Purified TSR and NHR proteins and RNA substrate were obtained.Both TSR and NHR proteins could methylate RNA substrate,and NHR protein showed better activity;both TSR and NHR proteins could bind with RNA substrate,and the binding intensity enhanced with the protein concentration. Conclusions Both TSR and NHR proteins can methylate the 1067A site of 23S rRNA,and induce antibiotics resistance of bacteria.

关 键 词：抗生素耐药性 23S rRNA甲基转移酶 硫链丝菌肽 那西肽 

分 类 号：Q555.5[生物学—生物化学]