Domain evolution in the GH13 pullulanase subfamily with focus on the carbohydrate-binding module family 48

Glycoside hydrolase (GH) family 13 comprises about 30 different specificities. Four of them have been proposed to form the GH13 pullulanase subfamily: pullulanase, isoamylase, maltooligosyl trehalohydrolase and branching enzyme forming the seven CAZy GH13 subfamilies: GH13 8-GH13 14. Recently, a new family of carbohydrate-binding modules (CBMs), the family CBM48 has been established containing the putative starch-binding domains from the pullulanase subfamily, the beta-subunit of AMP-activated protein kinase and some other GH13 enzymes with pullulanase and/or alpha-amylase-pullulanase specificity. Since all of these enzymes are multidomain proteins and the structure for at least one representative of each enzyme specificity has already been determined, the main goal of the present study was to elucidate domain evolution within this GH13 pullulanase subfamily (84 real enzymes) focusing on the CBM48 module. With regard to CBM48 positioning in the amino acid sequence, the N-terminal end of a protein appears to be a predominant position. This is especially true for isoamylases and maltooligosyl trehalohydrolases. Secondary structure-based alignment of CBM modules from CBM48, CBM20 and CBM21 revealed that several residues known as consensus for CBM20 and CBM21 could also be identified in CBM48, but only branching enzymes possess the aromatic residues that correspond with the two tryptophans forming the evolutionary conserved starch-binding site 1 in CBM20. The evolutionary trees constructed for the individual domains, complete alignment, and the conserved sequence regions of the alpha-amylase family were found to be comparable to each other (except for the C-domain tree) with two basic parts: (i) branching enzymes and maltooligosyl trehalohydrolases; and (ii) pullulanases and isoamylases. Taxonomy was respected only within clusters with pure specificity, i. e. the evolution of CBM48 reflects the evolution of specificities rather than evolution of species. This is a feature different from the one observed for the starch-binding domain of the family CBM20 where the starch-binding domain evolution reflects the evolution of species.