No abstract

Human maltase-glucoamylase (MGAM) and sucrase-isomaltase (SI) are small intestinal enzymes that work concurrently to hydrolyze the mixture of linear ␣-1,4-and branched ␣-1,6-oligosaccharide substrates that typically make up terminal starch digestion products. MGAM and SI are each composed of duplicated catalytic domains, N-and C-terminal, which display overlapping substrate specificities. The N-terminal catalytic domain of human MGAM (ntMGAM) has a preference for short linear ␣-1,4-oligosaccharides, whereas N-terminal SI (ntSI) has a broader specificity for both ␣-1,4-and ␣-1,6-oligosaccharides. Here we present the crystal structure of the human ntSI, in apo form to 3.2 Å and in complex with the inhibitor kotalanol to 2.15 Å resolution. Structural comparison with the previously solved structure of ntMGAM reveals key active site differences in ntSI, including a narrow hydrophobic ؉1 subsite, which may account for its additional substrate specificity for ␣-1,6 substrates.In humans, six enzyme activities (two ␣-amylase and four ␣-glucosidase activities) are involved in the breakdown of dietary starches and sugars into glucose. The ␣-glucosidase activities are associated with two small intestinal membrane-bound enzymes: maltase-glucoamylase (MGAM) 3 and sucrase-isomaltase (SI) (for a review, see Refs. 1 and 2). MGAM and SI are composed of duplicated catalytic domains: an N-terminal membrane-proximal domain (ntMGAM and ntSI) and a C-terminal luminal domain (ctMGAM and ctSI). The domains are anchored to the small intestinal brush-border membrane via an O-glycosylated stalk stemming from the N-terminal domain. Given that MGAM and SI genes arose from duplication and divergence of an ancestral gene, which itself has undergone tandem duplication (3), the N-terminal domains of MGAM and SI are more similar to one another in sequence, as are the C-terminal domains (ϳ60% sequence identity), than are the N-and C-terminal domains associated with the same enzyme (ϳ40% sequence identity).Within the carbohydrate-active enzymes (CAZY) classification system (36), which groups enzymes based on sequence similarity and reflects the functional and structural similarities of family members, N-and C-terminal MGAM and SI domains are members of the glycoside hydrolase 31 family (GH31). The four domains exhibit exo-glucosidase activities against ␣-1,4-linked maltose substrates (Fig. 1A) but display different specificities for malto-oligosaccharides of various lengths (4 -6). ntSI and ctSI subunits have additional activity for the ␣-1,6 linkages of starch branch points (and isomaltose substrates; Fig. 1A) and the ␣-1,2 linkage of sucrose, respectively (7), and are historically referred to as isomaltase and sucrase.As they are involved in the breakdown of dietary sugars and starches, MGAM and SI are attractive targets for inhibition by ␣-glucosidase inhibitors as a means of controlling blood glucose levels in individuals with type 2 diabetes (8). Acarbose (Fig. 1B) is the most widely used ␣-glucosidase inhibitor currently on the market and h...

An approach to controlling blood glucose levels in individuals with type 2 diabetes is to target alpha-amylases and intestinal glucosidases using alpha-glucosidase inhibitors acarbose and miglitol. One of the intestinal glucosidases targeted is the N-terminal catalytic domain of maltase-glucoamylase (ntMGAM), one of the four intestinal glycoside hydrolase 31 enzyme activities responsible for the hydrolysis of terminal starch products into glucose. Here we present the X-ray crystallographic studies of ntMGAM in complex with a new class of alpha-glucosidase inhibitors derived from natural extracts of Salacia reticulata, a plant used traditionally in Ayuverdic medicine for the treatment of type 2 diabetes. Included in these extracts are the active compounds salacinol, kotalanol, and de-O-sulfonated kotalanol. This study reveals that de-O-sulfonated kotalanol is the most potent ntMGAM inhibitor reported to date (K(i) = 0.03 microM), some 2000-fold better than the compounds currently used in the clinic, and highlights the potential of the salacinol class of inhibitors as future drug candidates.