Hibi Takao, Imaoka Masashi, Shimizu Yoichiro, Itoh Takafumi, Wakayama Mamoru
a Department of Bioscience and Biotechnology , Fukui Prefectural University , Fukui , Japan.
b Department of Biotechnology , College of Life Sciences, Ritsumeikan University , Kusatsu, Shiga , Japan.
Biosci Biotechnol Biochem. 2019 Feb;83(2):262-269. doi: 10.1080/09168451.2018.1547104. Epub 2018 Dec 3.
Theanine (γ-glutamylethylamide) is an amino acid analog that reduces blood pressure and improves immune responses. The ϒ-glutamyltranspeptidase (GGT) from Pseudomonas nitroreducens IFO12694 (PnGGT) has a unique preference for primary amines as ϒ-glutamyl acceptors over standard L-amino acids and peptides. This characteristic is useful for the synthesis of theanine. We used X-ray crystallographic analysis to understand the structural basis of PnGGT's hydrolysis and transpeptidation reactions and to characterize its previously unidentified acceptor site. Structural studies of PnGGT have shown that key interactions between three residues (Trp385, Phe417, and Trp525) distinguish PnGGT from other GGTs. We studied the roles of these residues in the distinct biochemical properties of PnGGT using site-directed mutagenesis. All mutants showed a significant decrease in hydrolysis activity and an increase in transpeptidase activity, suggesting that the aromatic side chains of Trp385, Phe417, and Trp525 were involved in the recognition of acceptor substrates. Abbreviations: ϒ-glutamyl peptide, theanine, X-ray crystallography.
茶氨酸（γ-谷氨酰乙酰胺）是一种氨基酸类似物，具有降低血压和改善免疫反应的作用。来自还原硝基假单胞菌IFO12694（PnGGT）的γ-谷氨酰转肽酶对伯胺作为γ-谷氨酰受体的偏好性独特，优于标准的L-氨基酸和肽。这一特性对茶氨酸的合成很有用。我们利用X射线晶体学分析来了解PnGGT水解和转肽反应的结构基础，并对其先前未确定的受体位点进行表征。PnGGT的结构研究表明，三个残基（Trp385、Phe417和Trp525）之间的关键相互作用使PnGGT区别于其他GGT。我们使用定点诱变研究了这些残基在PnGGT独特生化特性中的作用。所有突变体的水解活性均显著降低，转肽酶活性增加，这表明Trp385、Phe417和Trp525的芳香侧链参与了受体底物的识别。缩写：γ-谷氨酰肽、茶氨酸、X射线晶体学。
