Discovery and characterization of temperature adaptation modules by mining L-glutamate decarboxylase derived from psychrophilic microorganisms.

Directed alterations of the enzyme's temperature adaptations, such as thermostability and optimal catalytic temperature (T), are usually the way to go in order to meet the applications, however they are limited by the understanding between protein structure and function. Glutamate decarboxylase (GADs) is a common enzyme in the food industry. In this study, we identified and characterized three novel L-glutamate decarboxylases from psychrophilic microorganisms. Interestingly, the two enzymes (Gad Psy.Y/Gad Psy.F) showed significant differences in temperature adaptation. Through multiple sequence alignment and structural analysis, two potential regions that may affect the T and thermostability of the GAD, termed temperature adaptation modules (TAMs), were targeted. To validate the role of TAMs, we constructed mutants with bi-directional transplants of TAMs, which ultimately significantly altered the temperature adaptation of Gad Psy.Y and Gad Psy.F. Molecular dynamics simulations analysis demonstrated that the introduction and disruption of salt bridges in the mutant TAMs are crucial for this alteration. These findings deepen the understanding of the mechanisms of protein temperature adaptation and provide implications for protein engineering, while also offering advantageous enzymes to support the biosynthesis of GABA.