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Vol 57(2023) N 2 p. 204-213; DOI 10.1134/S0026893323020218 Full Text

T.E. Tyugashev1, O.S. Fedorova1, N.A. Kuznetsov1*

Modern Approaches to Protein Engineering to Create Enzymes with New Catalytic Properties

1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Novosibirsk, 630090 Russia

*nikita.kuznetsov@niboch.nsc.ru
Received - 2022-08-08; Revised - 2022-09-16; Accepted - 2022-09-16

Adenine-DNA-glycosylase MutY is a monofunctional enzyme and catalyzes hydrolysis of N-glycosidic bonds with adenine residues located opposite 8-oxonuanine residues in DNA. Rational design was carried out to construct mutant enzyme forms with altered catalytic activity. Structures of the MutY mutants were calculated by molecular dynamics (MD). Their analysis showed that some of the MutY mutants may have AP lyase activity in addition to hydrolyzing the N-glycosidic bond, as is the case with bifunctional DNA glycosylases. MutY mutants with the A120K or S124K substitution were obtained by site-directed mutagenesis, and their catalytic activities were determined. The S120K substitution was shown to confer additional AP lyase activity, while the A124K substitution completely inactivated the enzyme.

biocatalysis, rational design of enzymes, adenine-DNA glycosylase, specificity, catalytic activity, conformational changes



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