ИСТИНА

Enzymes are natural catalysts, which are able to catalyze effectively synthetic and degradation reactions under various environmental conditions. Specificity, stereoselectivity and adaptation of enzymes to a wide range of reaction conditions inspired their applications for chemical and pharmaceutical industries. However, the applications of enzymes are confined by their substrate specificity and adaptations to the natural cell environment. Studies of structure-function relationships in enzymes lead to the improvement of approaches to the regulation of their catalytic properties for biotechnological tasks [1]. Transaminases (TAs) are promising biocatalysts for the synthesis of optically pure amines. D-amino acid transaminase (DAAT) from Aminobacterium colombiense (AmicoTA) catalyzes the stereoselective transfer of an amino group between D-amino acid and α-keto acid to produce new D-amino acid and α-keto acid. AmicoTA is a thermostable (works up to 60 °C) enzyme with a pH-optimum 8.5-9.5. AmicoTA differs from DAAT from Bacillus subtilis by the organization of the active site, which includes an alternative carboxylate trap formed by Arg27, Thr34, and His175 residues together with several positively charged residues. Using X-ray analysis, we identified residues that are responsible for the substrate binding. Using site-directed mutagenesis, kinetic and spectral analysis, molecular modeling, and X-ray analysis we revealed residues that determine the pH-profile and stability of the enzyme, we distinguished residues bearing only specificity function and polyfunctional residues. Using kinetic analysis and MD simulations, we observed different binding modes of substrates with various functional groups. The Lys237Ala substitution led to a shift in the pH optimum of the enzyme to the acidic region (pH 5), while maintaining the stability of the enzyme and the level of activity.