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Methylumbelliferyl-β-cellobioside ( MUF- G2) is a convenient fluorogenic substrate for certain β-glycoside hydrolases ( GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes ( GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF- G2 in the +1 subsite of Hypocrea jecorina Cel6 A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-β-cellobiosides [6-chloro-4-methyl- (Cl MUF); 6-chloro-4-trifluoromethyl- (ClF3 MUF); 4-phenyl- (Ph UF); 6-chloro-4-phenyl- (ClPh UF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6 A was 10-150 times higher than with MUF- G2, although it was still three orders of magnitude lower than with H. jecorina Cel7 B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while Cl MUF- G2 could be used for determination of kcat and KM for H. jecorina Cel6 A and Thermobifida fusca Cel6 A. Crystal structures of H. jecorina Cel6 A D221 A mutant soaked with the MUF-, Cl MUF- and ClPh UF-β-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF- G2. Database Structural data have been submitted to the Protein Data Bank under accession numbers pdb 4AU0, 4AX7, 4AX6 Structured digital abstract • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 • Cel6A and Cel6A bind by x-ray crystallography (View Interaction: 1, 2) [ABSTRACT FROM AUTHOR]

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with ?-mannosidase to produce in yeast a form of the Pompe disease enzyme ?-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of ?-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease. [ABSTRACT FROM AUTHOR]

An efficient asymmetric synthesis of isofagomine, based on a precursor containing three differentiated hydroxyl functions, is described. The side product in the key alkylation step is converted into (2S,3R,4R)-2,4-bis(hydroxymethyl)-3-hydroxypyrrolidine, a new C6 pyrrolidine azasugar, which inhibits α-glucosidase from yeast. [ABSTRACT FROM AUTHOR]

Xyloglucan endo-transglycosylases (XETs) are key enzymes involved in the restructuring of plant cell walls during morphogenesis. As members of glycoside hydrolase family 16 (GH16), XETs are predicted to employ the canonical retaining mechanism of glycosyl transfer involving a covalent glycosyl-enzyme intermediate. Here, we report the accumulation and direct observation of such intermediates of PttXET16 -34 from hybrid aspen by electrospray mass spectrometry in combination with synthetic "blocked" substrates, which function as glycosyl donors but are incapable of acting as glycosyl acceptors. Thus, GalGXXXGGG and GalGXXXGXXXG react with the wild-type enzyme to yield relatively stable, kinetically competent, covalent GalG-enzyme and GalGXXXG-enzyme complexes, respectively (Gal = Galβ(1→4), G = Glcβ(1→4), and X = Xylα(1→6)Glcβ(1→4)). Quantitation of ratios of protein and saccharide species at pseudo-equilibrium allowed us to estimate the free energy change (ΔG0)tor the formation of the covalent GalGXXXG-enzyme as 6.3-8.5 kJ/mol (1.5-2.0 kcal/mol). The data indicate that the free energy of the 18(1→4) glucosidic bond in xyloglucans is preserved in the glycosyl-enzyme intermediate and harnessed for religation of the polysaccharide in vivo. [ABSTRACT FROM AUTHOR]

The substrate specificity of the xyloglucanase Cel74A from Hypocrea jecorina ( Trichoderma reesei) was examined using several polysaccharides and oligosaccharides. Our results revealed that xyloglucan chains are hydrolyzed at substituted Glc residues, in contrast to the action of all known xyloglucan endoglucanases (EC 3.2.1.151). The building block of xyloglucan, XXXG (where X is a substituted Glc residue, and G is an unsubstituted Glc residue), was rapidly degraded to XX and XG ( kcat = 7.2 s−1 and Km = 120 µm at 37 °C and pH 5), which has only been observed before with the oligoxyloglucan-reducing-end-specific cellobiohydrolase from Geotrichum (EC 3.2.1.150). However, the cellobiohydrolase can only release XG from XXXGXXXG, whereas Cel74A hydrolyzed this substrate at both chain ends, resulting in XGXX. Differences in the length of a specific loop at subsite + 2 are discussed as being the basis for the divergent specificity of these xyloglucanases. [ABSTRACT FROM AUTHOR]

ß-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 ß-glucosidases from Hypocrea jecorina, HjCel3A and HjCel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations. HjCel3B had a 7-fold higher specificity for cellobiose than HjCel3A but greater tendency for glucose inhibition. Energy decomposition analysis indicated that cellobiose has relatively weak electrostatic interactions with binding site residues, allowing it to be easily displaced by glucose and free to inhibit other hydrolytic enzymes. HjCel3A is, thus, preferable as an industrial ß-glucosidase despite its lower activity caused by transglycosylation. This competing pathway to hydrolysis arises from binding of glucose or cellobiose at the product site after formation of the glycosyl-enzyme intermediate. MD simulations revealed that binding is facilitated by hydrophobic interactions with Trp-37, Phe-260, and Tyr-443. Targeting these aromatic residues for mutation to reduce substrate affinity at the product site would therefore potentially mitigate transglycosidic activity. Engineering improved variants of HjCel3A and other structurally similar ß-glucosidases would have a significant economic effect on enzymatic biomass conversion in terms of yield and production cost as the process can be consequently conducted at higher substrate loadings. [ABSTRACT FROM AUTHOR]

Reports on the establishment that the tunnel-shaped active site of Trichoderma reesei cellobiohydrolase Cel6A contains two aspartic acids, D221 and D175. Use of site-directed mutagenesis, X-ray crystallography and enzyme kinetic studies to confirm the role of residue D221 as the catalytic acid; Effect of D175 on the protonation of D221.