Bacterial γ-glutamyltranspeptidases, physiological function, structure, catalytic mechanism and application.

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Gamma-glutamyltranspeptidase (GGT) is a key enzyme marker for hepatic and biliary diseases.
• Despite extensive study, fundamental enzymatic characteristics of GGT remain unelucidated.
• Bacterial GGT offers a model for understanding conserved physiological functions and enzymatic mechanisms.

Purpose of the Study:

• To obtain and characterize homogeneous bacterial GGT preparations.
• To elucidate the physiological function of GGT common to mammalian cells.
• To determine the catalytic mechanism, structure, and post-translational processing of bacterial GGT.
• To explore applications of bacterial GGT in enzymatic synthesis and enzyme engineering.

Main Methods:

• Purification of homogeneous bacterial GGT.
• Characterization of enzymatic properties and physiological function using GGT-deficient E. coli.
• Identification of catalytic nucleophile and post-translational processing.
• X-ray crystallography for structure determination.
• Trapping of reaction intermediates for mechanism elucidation.
• Enzymatic synthesis of gamma-glutamyl compounds.
• Site-directed and random mutagenesis for enzyme engineering.

Main Results:

• Homogeneous bacterial GGTs were obtained and characterized.
• The physiological function of GGT, conserved in mammalian cells, was elucidated.
• The catalytic nucleophile, post-translational processing, and crystal structure of E. coli GGT were identified.
• A structure-based reaction mechanism was presented.
• Enzymatic synthesis of valuable gamma-glutamyl compounds was achieved.
• Salt-tolerant Bacillus subtilis GGT was identified as a potential glutaminase for the food industry.
• Bacterial GGT was successfully engineered into glutaryl-7-aminocephalosporanic acid acylase for antibiotic production.

Conclusions:

• Bacterial GGT provides crucial insights into fundamental enzymatic characteristics and conserved physiological functions.
• The elucidated structure and mechanism pave the way for novel applications.
• Engineered bacterial GGT holds significant potential in the food, nutraceutical, medicinal, and pharmaceutical industries.