Background: Newborn screening for deficiency in the lysosomal enzymes that cause Fabry, Gaucher, Krabbe, Niemann-Pick A/B, and Pompe diseases is warranted because treatment for these syndromes is now available or anticipated in the near feature. We describe a multiplex screening method for all five lysosomal enzymes that uses newborn-screening cards containing dried blood spots as the enzyme source. Methods: We used a cassette of substrates and internal standards to directly quantify the enzymatic activities, and tandem mass spectrometry for enzymatic product detection. Rehydrated dried blood spots were incubated with the enzyme substrates. We used liquid-liquid extraction followed by solid-phase extraction with silica gel to remove buffer components. Acarbose served as inhibitor of an interfering acid ␣-glucosidase present in neutrophils, which allowed the lysosomal enzyme implicated in Pompe disease to be selectively analyzed. Results: We analyzed dried blood spots from 5 patients with Gaucher, 5 with Niemann-Pick A/B, 11 with Pompe, 5 with Fabry, and 12 with Krabbe disease, and in all cases the enzyme activities were below the minimum activities measured in a collection of heterozygous carriers and healthy noncarrier individuals. The enzyme activities measured in 5-9 heterozygous carriers were approximately one-half those measured with 15-32 healthy individuals, but there was partial overlap of each condition between the data sets for carriers and healthy individuals. Conclusion: For all five diseases, the affected individuals were detected. The assay can be readily automated,

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...

The retaining glycosyltransferase GalNAc-T2 is a member of a large family of human polypeptide GalNAc-transferases that is responsible for the post-translational modification of many cell-surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum-mechanics/molecular-mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic-electronic level of detail. Our study provides a detailed structural rationale for an ordered bi-bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front-face SN i-type reaction in which the substrate N-acetyl sugar substituent coordinates efficient glycosyl transfer.

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.

UDP-galactopyranose mutase (UGM) is the key enzyme involved in the biosynthesis of Galf. UDPGalp and UDP-Galf are two natural substrates of UGM. A protocol that combines the use of STD-NMR spectroscopy, molecular modeling, and CORCEMA-ST calculations was applied to the investigation of the binding of UDP-Galf and its C3-fluorinated analogue to UGM from Klebsiella pneumoniae. UDP-Galf and UDP-[3-F]Galf were bound to UGM in a similar manner as UDPGalp. The interconversions of UDP-Galf and UDP-[3-F]Galf to their galactopyranose counterparts were catalyzed by the reduced (active) UGM with different catalytic efficiencies, as observed by NMR spectroscopy. The binding affinities of UDP-Galf and UDP-[3-F]Galf were also compared with those of UDP-Galp and UDP by competition STD-NMR experiments. When UGM was in the oxidized (inactive) state, the binding affinities of UDP-Galf, UDP-Galp, and UDP-[3-F]Galf were of similar magnitudes, and were lower than that of UDP. However, when UGM was in the reduced state, UDP-Galp had higher binding affinity compared with UDP. Molecular dynamics (MD) simulations indicated that the "open" mobile loop in UGM "closes" upon binding of the substrates. Combined MD simulations and STD-NMR experiments were used to create models of UGM with UDP-Galf and UDP-[3-F]Galf as bound ligands. Calculated values of saturation-transfer effects with CORCEMA-ST (complete relaxation and conformational exchange matrix analysis of saturation transfer) were compared to the experimental STD effects, and permitted differentiation between two main conformational families of the bound ligands. Taken together, these results are used to rationalize the different rates of catalytic turnover of UDP-Galf and UDP-[3-F]Galf, and shed light on the mechanism of action of UGM.