The fifth conserved sequence region as a selection marker distinguishing between oligo‐1,6‐glucosidase, neopullulanase, and intermediary groups of the α‐amylase family GH13

The α-amylase or glycoside hydrolase family 13 (GH13) is the largest in the CAZy database, containing ~239,000 members representing more than 30 enzyme specificities in 50 GH13 subfamilies. Knowledge of features in seven conserved sequence regions (CSRs) is helpful for in silico predictions preceding experimental correlation of enzyme specificity with a subfamily. Before GH13 got divided into subfamilies in CAZy, two large groups, called the oligo-1,6-glucosidase (OGLU) and neopullulanase (NPU) "subfamilies," were proposed as having the CSR-V QpDln and MpKln, respectively; MpDln was assigned to an intermediary (IM) group. As new GH13 subfamilies were defined, the original OGLU and NPU groups expanded. Therefore, an exhaustive bioinformatics analysis was conducted on ~72,000 sequences (almost one third of family GH13) covering the 16 subfamilies in the OGLU (GH13_4, 16, 17, 18, 23, 29, 30, 31, 35, 40, 44), NPU (GH13_20, 21 and 39), and IM (GH13_36 and 46) groups. Comparison of sequence logos of the seven CSRs for each group and for their individual subfamilies identified unique positions associated with specific subfamilies and/or specificities. Two unrooted phylogenetic trees, one using an alignment spanning from β2 through β8 of the catalytic TIM-barrel including domain B, and another calculated for the seven CSRs, revealed that new determinants may emerge. Moreover, a structure-stabilizing role was proposed for CSR-V, and structures and close functional and evolutionary relatedness of GH13_36 and GH13_46 are discussed. In summary, since highly specific CSR signatures, including of CSR-V, can occur in several subfamilies, they seem more connected with enzyme specificity than to a particular GH13 subfamily.