title: [Cloning, expression and characterization of the nucleotidylyltransferase gene-amiE in amicetin biosynthesis].
abstract: 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 Ni2+ 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Horticulture Science for Southern Mountainous Regions, Ministry of Education, College of Horticulture and Landscape Architecture, Southwest University, Chongqing 400715, China. hutao_122@126.com
authors.forename: Tao
authors.initials: T
authors.lastname: Hu
authors.forename: Guangtao
authors.initials: G
authors.lastname: Zhang
authors.forename: Yiguang
authors.initials: Y
authors.lastname: Zhu
authors.forename: Sumei
authors.initials: S
authors.lastname: Li
authors.forename: Haibo
authors.initials: H
authors.lastname: Zhang
authors.forename: Gaiyun
authors.initials: G
authors.lastname: Zhang
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Yang
authors.forename: Jianhua
authors.initials: J
authors.lastname: Ju
authors.forename: Changsheng
authors.initials: C
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics* / Q000502:physiology; D011741:Pyrimidine Nucleosides / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
chemical_list: D000900:Anti-Bacterial Agents; D011741:Pyrimidine Nucleosides; D011994:Recombinant Proteins; C004413:amicetin; D009713:Nucleotidyltransferases