Enzymatic activity verification and protein three-dimensional structural analysis of theanine synthetase gene in Camellia sinensis.

The aim of this study was to analyze the highly homologous sequences of theanine synthetase genes and glutamine synthetase genes of tea plant, speculate their evolutionary relationship, predict their structures, natures, functions and differences of enzyme active sites, and then to provide a basis for CsTS genes function studies of tea. Two gene sequences logged on NCBI were cloned, the expression of enzymes activities was verificated through HPLC and LC-MS and phylogenetic analysis was implemented within the searching result. The expression of enzymes in E. coli suggested CsTS and CsGS had different catalytic activities although their high homology. The primary and secondary protein structures of CsTS and CsGS were analyzed and the result showed that there was no significant difference between them, but the third protein structure analysis by homology modeling in the catalytic sites showed three differences in the catalytic sites, which could lead to the difference of enzyme activity. In this research, CsTS gene sequences were firstly identified as the members of GS gene family through phylogenetic analysis. According to cellular localization prediction, CsTS should belong to cytoplasmic GS(GS1), for its kinship close to grape, upland cotton, rubber tree, arabidopsis etc. [ABSTRACT FROM AUTHOR]