Modern Approaches of Protein Engineering for the Creation of Enzymes with New Catalytic Properties

Abstract

Adenine-DNA-glycosylase MutY is a monofunctional enzyme and catalyzes the hydrolysis of N-glycosidic bonds with adenine residues located opposite 8-oxonuanine residues in DNA. A rational design of mutant forms of the enzyme with altered catalytic activity was carried out. An analysis of the structures of mutant forms of MutY calculated by the molecular dynamics method led to the conclusion that some mutant forms of MutY, in addition to hydrolysis of the N-glycosidic bond, may have AP-lyase activity, as in the case of bifunctional DNA glycosylases. Mutant forms of MutY containing substitutions A120K or S124K were obtained by site-directed mutagenesis, and their catalytic activity was determined. It was shown that the S120K substitution leads to the appearance of additional AP-lyase activity, while the A124K substitution completely inactivates the enzyme.