Tracing the evolution of the α-amylase subfamily GH13_36 covering the amylolytic enzymes intermediate between oligo-1,6-glucosidases and neopullulanases.

Among the glycoside hydrolases (GHs) classified within the Carbohydrate-Active enZymes (CAZy) server, the α-amylase family GH13 belongs to the largest GH families. It has been divided into the official 36 subfamilies by the CAZy curators. Originally the subfamilies of oligo-1,6-glucosidase and neopullulanase were defined using the sequence of the fifth conserved sequence region (CSR) as a selection marker. It is localized outside the catalytic α-amylase (β/α)(8)-barrel in the domain B, that is, in a longer loop connecting the strand β3 with the helix α3 of the barrel. It is sequentially positioned 26-28 residues in front of the invariant aspartic acid residue in the β4-strand acting as the GH13 catalytic nucleophile. The CSR V is characteristic as QpDln and MpKln for the former and latter subfamilies, respectively. A group of intermediate sequences possessing the CSR V as a mix of the two above-mentioned subfamilies, that is, MpDln, was also proposed previously. The present bioinformatics analysis was done in an effort to reveal as many as possible GH13 members of this intermediary group, currently classified as the subfamily GH13_36, and to discuss their evolutionary relationships to known GH13 specificities as well as with regard to their taxonomic origin. Using the BLAST tool with the sequence of the α-amylase from Halothermothrix orenii AmyA exhibiting the intermediary features, 152 GH13 enzymes, and hypothetical proteins were retrieved covering defined specificities (GH13 subfamilies 4, 16, 17, 18, 20, 21, 23, 29, 30, 31, 34, and 35) and intermediary enzymes and proteins (GH13_36). In both evolutionary trees-based on the alignment of CSRs and complete sequences-most of the 'intermediary' proteins (i.e., those with MPDLN signature) were positioned in several closely related clusters forming, however, a single GH13_36 large part of the trees. A few novel GH13 subfamilies were proposed as well as the specificity implications were discussed based on the presented in silico analysis. The results may also be helpful in assigning any GH13-like amino acid sequence the subfamily GH13_36 affiliation without additional biochemical characterization.