友菌素核苷转移酶amiE基因的克隆、表达和功能鉴定

【目的】克隆和表达二糖核苷类抗生素友菌素生物合成基因簇中的核苷转移酶基因amiE,并研究AmiE的体外催化功能。【方法】采用PCR技术将编码257个氨基酸的葡萄糖-1-磷酸核苷转移酶基因amiE克隆到表达载体pET28a上,构建质粒pCSG4001,转化入大肠杆菌E.coli BL21(DE3)中诱导表达;利用亲和层析分离纯化蛋白AmiE,以葡萄糖-1-磷酸和胸腺嘧啶三磷酸(TTP)或尿嘧啶三磷酸(UTP)为底物,利用高效液相检测AmiE的体外酶活;以甘露糖-1-磷酸、半乳糖胺-1-磷酸和半乳糖-1-磷酸和TTP作为底物,进一步研究AmiE对底物的选择性。【结果】N-末端融合组氨酸标签的AmiE蛋白在大肠杆菌中获得了可溶性表达,通过亲和层析纯化出的AmiE能够以TTP(或UTP)和葡萄糖-1-磷酸作为底物,催化形成胸腺嘧啶二磷酸葡萄糖(TDP-glucose)或者尿嘧啶二磷酸葡萄糖(UDP-glucose),但对其他三种底物,无明显催化活性。【结论】大肠杆菌中表达纯化的核苷转移酶AmiE能够体外催化形成TDP-葡萄糖(或UDP-葡萄糖),确证了AmiE作为核苷转移酶的催化功能,同时表明AmiE对底物具有一定的选择性。

Cloning, expression and characterization of the nucleotidylyltransferase gene-amiE in amicetin biosynthesis

Objective]The aim of this study is to clone and express the nucleotidylytransferase encoding gene-amiE from the biosynthetic gene cluster of amicetin,a disaccharide nucleoside antibiotic,and to characterize AmiE in vitro.Methods]The amiE,encoding a nucleotidylytransferase of 257 amino acid,was PCR amplified and cloned into pET28a,resulting in the plasmid pCSG4001,which was transformed into E.coli BL21(DE3) for expressing N-(His)6-tag AmiE.The recombinant AmiE was purified by affinity chromatography via AKTA Purifier 10 system.The AmiE-catalyzed reactions were performed using TTP(or UTP) and glucose-1-phosphate as substrates.The enzyme assays were analyzed by HPLC;the substrate flexibility of AmiE was probed with three unnatural sugars-1-phosphate,including galactose-1-phosphate,galactosamine-1-phosphate and mannos-1-phosphate.Results]The N-(His)6-tag AmiE was expressed in E.coli in soluble form and was successfully purified via Ni+2 mediated affinity chromatography;in vitro biochemical experiments showed that AmiE could convert glucose-1-phosphate into TDP-glucose(or UDP-glucose) in the presence of TTP(or UTP).However,galactose-1-phosphate,galactosamine-1-phosphate and mannos-1-phosphate were not substrates of AmiE.Conclusion]The amiE was successfully cloned and expressed in E.coli,and the purified AmiE was biochemically confirmed to be a nucleotylyltransferase in amicetin biosynthesis pathway.