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Comparative bioinformatics is the cornerstone of computational approaches to understanding structure-functional relationship in enzymes. The members of α/β hydrolase-fold superfamily represent a functionally diverse group of enzymes with common structural organization that appear to have lost sequence similarity during natural selection and specialization from a common ancestor. At the same time their active site structures in general remain conserved while other parts may largely differ. It is therefore expected that three-dimensional alignment will provide more significant clues to protein function, properties and evolution than sequence alignment alone. Here we report the largest so far multiple structural comparison of α/β hydrolase-fold superfamily enzymes and analysis of catalytically important residues considering distribution of amino acid types among enzymes with different catalytic properties. A computer algorithm has been developed for high-throughput structural comparison of homologous enzymes. Comparative analysis of the most functionally significant parts of enzyme structures – the active sites – is suggested as a source of new understanding of structure-functional relationship in α/β hydrolases. On the first step a library of active site structures of α/β hydrolases – amino acids involved in catalysis together with residues forming the active site cavity and thus potentially involved in mechanical aspects of enzyme behavior by interacting with the substrate or catalytic machinery – was created using previously reported procedure [1]. Then a representative set of structures as the basis for comparison of distinct subfamilies was selected using 95% sequence similarity threshold. On the next step a superimposition matrix was created from pairwise comparisons of representative structures. Finally, amino acid positions conserved between the structures were determined and clustered to form the common core alignment. Multiple alignment of α/β-hydrolase superfamily was created on the basis of 238 non-redundant structure set of enzymes with lipase, esterase, hydroxynitrile lyase, epoxide hydrolase, peptidase, dienelactone hydrolase and dehalogenase activities. To the best of our knowledge this is the largest structural alignment of the superfamily reported so far. Comparative analysis revealed a major structural similarity of active site regions while the most significant fit was observed near the catalytic triad residues. While catalytic His was found to be conserved among all α/β-hydrolase enzymes, the nucleophile and catalytic acid were identified as subfamily-specific positions - residues with a tendency to be conserved only within subfamilies of enzymes, but different between subfamilies. Nucleophile position can be occupied by Ser, Asp or Cys. Ser was found to be common for the majority of activities explored. Asp is considered as stronger nucleophile compared to Ser and contributes to SN2 reaction mechanism of epoxide hydrolases and dehalogenases. Dienelactone hydrolases have Cys as a nucleophile due to substrate-assisted catalysis, where a functional group in the substrate controls the protonation state of the nucleophile. Catalytic acid was found to be represented by Asp or Glu. Asp was shown to be the most common in this position, while Glu was found in acetylcholine esterases and some carboxypeptidases. Role of this substitution yet remains unknown. It was shown, that the origin of catalytic acids is not always homologous – in the majority of structures it is hosted by β7 sheet, though in 15% of cases it can be found on β6 sheet of the α/β-fold. Proteins with α/β-hydrolase fold that do not have reported enzymatic activity like gibberellin receptors, cell adhesion proteins, signaling proteins etc. were also considered. It was shown that catalytic triad positions are occupied by residues that do not support catalytic activity, like Gly in nucleophile position or Val in place of a catalytic histidine.