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Enzymes within single family usually share a common function but differ in more specific features and can be divided into subfamilies with different specificity, enantioselectivity, stability, etc. New bioinformatic analysis methodology has been developed to identify function-related variable residues in protein structures that are responsible for functional divergence within families of homologous enzymes. We suggest using a term “subfamily-specific position(s)” or SSP(s) to outline those residues to be conserved within subfamilies of enzymes, but different between subfamilies. Specificity scores are ranked taking into account structural information alongside with sequences, hits are chosen based on assigned statistical significance. Bioinformatic analysis of alpha-beta hydrolase enzyme superfamily was performed. Multiple structure-guided sequence alignment was created based on 238 alpha-beta hydrolase PDB structures. Bioinformatic analysis revealed SSPs responsible for discrimination between lipase-amidase activities and esterase-hydroxynitrile lyase activities within alpha-beta hydrolase fold. Common structural organization of totally conserved positions of catalytic residues and oxyanion holes was observed among serine carboxypeptidase, lipase B and hydroxynitrile lyase despite significant difference of functional properties and ability to catalyze distinct chemical transformations. Developed methodology was also applied to study evolution of structure-functional relationship in other enzyme families: Ntn-hydrolases, penicillin-binding proteins, etc. It was shown, that patterns of SSPs can be effectively used to design enzyme mutants with improved catalytic properties and to predict functional properties of newly discovered enzymes.