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PMID:19966457

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Citation

Miyake, K and Kakita, S (2009) A novel catalytic ability of gamma-glutamylcysteine synthetase of Escherichia coli and its application in theanine production. Biosci. Biotechnol. Biochem. 73:2677-83

Abstract

Gamma-glutamylcysteine synthetase (gammaGCS, EC 6.3.2.2) catalyzes the formation of gamma-glutamylcysteine from L-glutamic acid (Glu) and L-cysteine (Cys) in an ATP-dependent manner. While gammaGCS can use various amino acids as substrate, little is known about whether it can use non-amino acid compounds in place of Cys. We determined that gammaGCS from Escherichia coli has the ability to combine Glu and amines to form gamma-glutamylamides. The reaction rate depended on the length of the methylene chain of the amines in the following order: n-propylamine > butylamine > ethylamine > methylamine. The optimal pH for the reaction was narrower and more alkaline than for the reaction with an amino acid. The newly found catalytic ability of gammaGCS was used in the production of theanine (gamma-glutamylethylamine). The resting cells of E. coli expressing gammaGCS, in which ATP was regenerated through glycolysis, synthesized 12.1 mM theanine (18 h) from 429 mM ethylamine.

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Keywords

Biocatalysis; Escherichia coli/cytology; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/metabolism; Ethylamines/metabolism; Glutamate-Cysteine Ligase/biosynthesis; Glutamate-Cysteine Ligase/isolation & purification; Glutamate-Cysteine Ligase/metabolism; Glutamates/biosynthesis; Hydrogen-Ion Concentration; Substrate Specificity

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