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

Abstract

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 β-subunit of AMP-activated protein kinase and some other GH13 enzymes with pullulanase and/or α-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 α-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.

Microarray analysis of differential gene expression elicited in Trametes versicolor during interspecific mycelial interactions

“…Three probes encoded enzymes involved in carbohydrate metabolism including an a-amylase family (glycoside hydrolase family 13) gene and a 1,3-beta-glucan synthase. Glycoside hydrolase family 13 is a family of starch modifying enzymes comprising around 30 different specificities, including isoamylases, glucan synthases, and pullunases (Machovic & Janecek 2008). Recent studies have indicated that some GH13 family enzymes in fungi may be involved in the synthesis or modification of alpha glucan in the fungal cell wall rather than starch degradation (Yuan et al 2008).…”

Crystal Structure of an Essential Enzyme in Seed Starch Degradation: Barley Limit Dextrinase in Complex with Cyclodextrins

“…It precedes the catalytic (β/α) 8 -barrel-like domain. While LD and isoamylase have one CBM annotated as CBM48, 17 KpPUL and Bacillus acidopullulyticus pullulanase (BaPUL) contain both CBM41 and CBM48 domains. 18,19 All the endo α-1,6 glucoside hydrolases have a catalytic (β/α) 8 -like domain.…”

Deriving Chemically Essential Interactions Based on Active Site Alignments and Quantum Chemical Calculations: A Case Study on Glycoside Hydrolases

“…48,49 It together with the residues 4-D and 5-E has been named a catalytic triad. 50 From the aligned active sites here, the main role of residue 7-D seems to be not a chemical one but to hold the sugar ring in position through hydrogen bonding with the 2-hydroxyl. Residue 4-R was mostly conserved, but was replaced by Lys in some GH-77 proteins.…”