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Computational enzyme engineering has emerged as a powerful fast-growing segment of biotechnology. Despite certain success in the field a gap between success stories reported on individual examples and a high throughput methodology unified for effective use by the community indicates our limited understanding of structure-functional framework. Bioinformatic analysis of functionally important sites that are subject to regular subfamily-specific variations inside protein family is suggested as a generalized strategy for systematic protein design by comparative analysis of numerous sequences and structures deposited in databases. 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. The method has been implemented in a program Zebra for efficient bioinformatic analysis of substantial volumes of sequence and structural data. 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 method was also applied to study evolution of structure-functional relationship in Ntn-hydrolases and penicillin-binding proteins. Subfamily specific positions are evolutionary flexible and variable in nature to improve or change enzymatic functions and seem to be a good explanation for protein evolution. 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.