We have determined the first structure of a family 31 ␣-glycosidase, that of YicI from Escherichia coli, both free and trapped as a 5-fluoroxylopyranosyl-enzyme intermediate via reaction with 5-fluoro-␣-D-xylopyranosyl fluoride. Our 2.2-Å resolution structure shows an intimately associated hexamer with structural elements from several monomers converging at each of the six active sites. Our kinetic and mass spectrometry analyses verified several of the features observed in our structural data, including a covalent linkage from the carboxylate side chain of the identified nucleophile Asp 416 to C-1 of the sugar ring. Structure-based sequence comparison of YicI with the mammalian ␣-glucosidases lysosomal ␣-glucosidase and sucrase-isomaltase predicts a high level of structural similarity and provides a foundation for understanding the various mutations of these enzymes that elicit human disease.Glycoside hydrolases play critical roles in biology ranging from digestion and decomposition of polysaccharides to biosynthesis of glycoproteins. Gene sequences of many thousands of these important enzymes have now been determined, and the corresponding enzymes have been grouped into families on the basis of sequence similarity (1-5). 1The ␣-glucosidases are a particularly important subset of these enzymes, both in primary metabolism and in glycoconjugate biosynthesis and processing. These enzymes are principally found in families 13 and 31 and, to a lesser extent, in families 4 and 63. Family 13 contains a wide range of glucosideprocessing enzymes, including the ␣-amylases and cyclodextrin glucanotransferases. Correspondingly, enzymes of this family have attracted considerable attention, with numerous mechanistic studies and with three-dimensional structures having been known for some 20 years. By contrast, family 31 has received relatively little attention despite its importance and the number of different activities represented from a range of organisms, including animals, plants, and microorganisms (6, 7). This family contains such important ␣-glucosidases as the human lysosomal ␣-glucosidase, a deficiency of which results in Pompe's disease (also known as glycogen storage disease type 2 or acid maltase deficiency, the marked feature of which is the accumulation of glycogen in heart and skeletal muscle cells); the endoplasmic reticulum glucosidase II, which plays a key role in glycoprotein processing and folding; and the digestive enzyme sucrase-isomaltase, which is the target of inhibition by the anti-diabetes drugs acarbose and miglitol. It also contains ␣-xylosidases, isomaltosyltransferases, and the mechanistically interesting ␣-glucan lyases, which carry out an elimination reaction rather than hydrolysis.Despite the importance of this family, mechanistic insights are limited. The enzymes are known to be retaining ␣-glycosidases, which hydrolyze the glycosidic bond with net retention of anomeric configuration via an acid/base-catalyzed mechanism involving a covalent glycosyl-enzyme intermediate. Through a range of st...

New proteomics methods are required for targeting and identification of subsets of a proteome in an activity-based fashion. Here, we report the first gel-free, mass spectrometry-based strategy for mechanism-based profiling of retaining ␤-endoglycosidases in complex proteomes. Using a biotinylated, cleavable 2-deoxy-2-fluoroxylobioside inactivator, we have isolated and identified the active-site peptides of target retaining ␤-1,4-glycanases in systems of increasing complexity: pure enzymes, artificial proteomes, and the secreted proteome of the aerobic mesophilic soil bacterium Cellulomonas fimi. The active-site peptide of a new C. fimi ␤-1,4-glycanase was identified in this manner, and the peptide sequence, which includes the catalytic nucleophile, is highly conserved among glycosidase family 10 members. The glycanase gene (GenBank TM accession number DQ146941) was cloned using inverse PCR techniques, and the protein was found to comprise a catalytic domain that shares ϳ70% sequence identity with those of xylanases from Streptomyces sp. and a family 2b carbohydrate-binding module. The new glycanase hydrolyzes natural and artificial xylo-configured substrates more efficiently than their cello-configured counterparts. It has a pH dependence very similar to that of known C. fimi retaining glycanases.With the completion of the genome sequences of many organisms, the field of proteomics faces several major tasks. One challenge is that of identification and assignment of structure/function to the tens of thousands of proteins encoded by prokaryotic and eukaryotic genomes. Another challenge is accurate quantitative analysis of changes in protein levels/activities that occur within a proteome as a response to biological perturbations that are due either to normal developmental and metabolic changes or to abnormalities associated with disease. Proteomic techniques such as comparative two-dimensional gel electrophoresis coupled with mass spectrometry, the isotope-coded affinity tagging approach (1), and variations of isotope-coded affinity tagging that identify sites of modification (e.g. glycosylation and phosphorylation) on proteins (2-4) fail to provide a direct assessment of protein function.Recently, several chemical strategies for activity-based protein profiling (ABPP) 4 in complex proteomes that target several enzyme groups, including oxidoreductases (5, 6), serine hydrolases (7, 8), cysteine proteases (9, 10), threonine proteases (11), metalloproteases (12), protein phosphatases (13), kinases (14), and exoglycosidases (15), have been employed. ABPP probes have two general features: 1) an active sitedirected (mechanism-based) inactivator or affinity label that reacts with a catalytic residue and forms a covalent adduct with the target enzyme(s) and 2) one or more reporter groups that enable rapid detection (e.g. a fluorophore) and/or affinity isolation (e.g. biotin) (16). As such, ABPP methods can provide direct information on post-translational forms of protein regulation (17). However, most of the ABPP research ...

The Agrobacterium sp. ␤-glucosidase (Abg) is a retaining ␤-glycosidase and its nucleophile mutants, termed Abg glycosynthases, catalyze the formation of glycosidic bonds using ␣-glycosyl fluorides as donor sugars and various aryl glycosides as acceptor sugars. Two rounds of random mutagenesis were performed on the best glycosynthase to date (AbgE358G), and transformants were screened using an on-plate endocellulase coupled assay. Two highly active mutants were obtained, 1D12 (A19T, E358G) and 2F6 (A19T, E358G, Q248R, M407V) in the first and second rounds, respectively. Relative catalytic efficiencies (k cat /K m ) of 1:7:27 were determined for AbgE358G, 1D12, and 2F6, respectively, using ␣-D-galactopyranosyl fluoride and 4-nitrophenyl ␤-D-glucopyranoside as substrates. The 2F6 mutant is not only more efficient but also has an expanded repertoire of acceptable substrates. Analysis of a homology model structure of 2F6 indicated that the A19T and M407V mutations do not interact directly with substrates but exert their effects by changing the conformation of the active site. Much of the improvement associated with the A19T mutation seems to be caused by favorable interactions with the equatorial C2-hydroxyl group of the substrate. The alteration of torsional angles of Glu-411, Trp-412, and Trp-404, which are components of the aglycone (؉1) subsite, is an expected consequence of the A19T and M407V mutations based on the homology model structure of 2F6.Oligosaccharides have considerable potential as therapeutics because of the numerous medicinally relevant physiological events that involve glycoconjugates (1-3). To expand our understanding of the various roles of oligosaccharides found in important cellular events, more efficient and selective synthetic protocols must be developed for the preparation of oligosaccharides. Classical chemical synthesis is often impractical for the synthesis of complex oligosaccharides because of the need for selective and labor-intensive protection-deprotection steps and difficulties in directing product stereochemistry. To overcome these limitations, enzymatic syntheses using glycosidases or glycosyl transferases have rapidly gained prominence (4 -6).In recent years, the glycosynthase approach developed in this laboratory has added a new dimension to the enzymatic preparation of oligosaccharides (7-9). Glycosynthases are retaining glycosidase mutants in which the catalytic nucleophile has been converted to a non-nucleophilic residue. These mutants catalyze the formation of glycosidic bonds when glycosyl fluorides with anomeric configuration opposite to that of the original substrate, thereby mimicking the glycosyl enzyme intermediate, are employed as substrates. The modified enzyme catalyzes the nucleophilic displacement of the fluoride via attack by a hydroxyl group on an added glycosyl acceptor, generating a new glycosidic bond with the same stereochemistry as the normal substrate. The reactions catalyzed by glycosynthases are highly amenable to industrial syntheses because of the hig...

A gene encoding a maltogenic amylase of Bacillus stearothermophilus ET1 was cloned and expressed in Escherichia coli. DNA sequence analysis indicated that the gene could encode a 69627-Da protein containing 590 amino acids. The predicted amino acid sequence of the enzyme shared 47Ϫ70% identity with the sequences of maltogenic amylase from Bacillus licheniformis, neopullulanase from B. stearothermophilus, and cyclodextrin hydrolase (CDase) I-5 from an alkalophilic Bacillus I-5 strain. In addition to starch, pullulan and cyclodextrin, B. stearothermophilus could hydrolyze isopanose, but not panose, to glucose and maltose. Maltogenic amylase hydrolyzed acarbose, a competitive inhibitor of amylases, to glucose and a trisaccharide. When acarbose was incubated with 10% glucose, isoacarbose, containing an A-1,6-glucosidic linkage was produced as an acceptor reaction product. B. stearothermophilus maltogenic amylase shared four highly similar regions of amino acids with several amylolytic enzymes. The β-cyclodextrinϪhydrolyzing activity of maltogenic amylase was enhanced to a level equivalent to the activity of CDase when its amino acid sequence between the third and the fourth conserved regions was made more hydrophobic by site-directed mutagenesis. Enhanced transglycosylation activity was observed in most of the mutants. This result suggested that the members of a subfamily of amylolytic enzymes, including maltogenic amylase and CDase, could share similar substrate specificities, enzymatic mechanisms and structure/function relationships.Keywords : Bacillus stearothermophilus; maltogenic amylase ; acarbose ; transglycosylation; site-directed mutagenesis.Many types of amylases with unique properties have been megaterium [7], and A-amylase of Thermoactinomyces vulgaris [8] have been reported to hydrolyze the A-1,4 linkages of pulluisolated and characterized for various applications in the starch industry [1,2]. These proteins share many structural and mecha-lan to produce panose. Amylolytic enzymes, such as cyclodextrin glucanotransferases (CGTase) and CDase exhibit their nistic characteristics. However, amylases can be divided into several groups according to substrate specificities, patterns of highest levels of activity on cyclomaltodextrins [9Ϫ11]. Some starch cleavage, transglycosylation or cyclization activities, and amylolytic enzymes, including debranching enzymes and structural features. Classical A-amylases (e.g. 1,4-A-D-glucan CGTase, catalyze transglycosylation by forming A-1,4 or A-1,6 glucanohydrolase) catalyze hydrolysis of A-1,4-glucosidic link-linkages. ages in starch, and different amylases give rise to oligosacchaJespersen et al.[12] used sequence alignments and structurerides with specific lengths of glucose as major product [2]. De-prediction models to predict the presence of A-amylase-type branching enzymes are capable of hydrolyzing A-1,6-glucosidic (β/A) 8 -barrel domains and the positions of the β-strands and Alinkages in starch and/or pullulan [1, 3Ϫ5] to produce maltotri-helices found in 47 amy...

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