Domain evolution in enzymes of the neopullulanase subfamily.

Abstract

Among the glycoside hydrolases (GHs) classified within the Carbohydrate-Active enZyme (CAZy) database, the α-amylase family GH13 containing ~30 different enzyme specificities and more than 37 000 sequences represents one of the largest GH families. Earlier, based on a characteristic sequence motif in their fifth conserved sequence region, the two closely related subfamilies, the so-called oligo-1,6-glucosidase and neopullulanase subfamilies, were described. Currently, the two subfamilies cover several CAZy-defined GH13 subfamilies because the α-amylase family GH13 has officially been divided into 41 subfamilies. The subfamily GH13_20 also contains, in addition to neopullulanase, cyclomaltodextrinase and maltogenic amylase. These usually possess the N-terminal starch-binding domain (SBD) classified as the carbohydrate-binding module family CBM34. The present in silico study has been focused on the neopullulanase subfamily in an effort to shed some light on the evolution of its modular arrangement. The main goal was to reveal the evolutionary relationships between the catalytic domain representing the enzyme specificity and the non-catalytic SBDs. The studied set based on the CAZy subfamily GH13_20 and family CBM34 was completed by related amylolytic enzymes, such as α-amylases, glycogen debranching enzymes and amylopullulanases. It finally consisted of 74 mostly biochemically characterized GH13 enzymes. The analysed sequences were divided into nine groups based on the presence of various carbohydrate-binding module domains (CBM20 and CBM48 in addition to CBM34). A special unique domain arrangement was revealed in the the α-amylase from Bacillus sp. AAH-31, in which the three consecutive SBDs (i.e. CBM20, CBM48 and CBM34, in that order) are present at its N-terminus.