Using ab initio metadynamics we have computed the conformational free energy landscape of beta-D-glucopyranose as a function of the puckering coordinates. We show that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The number of free energy minima (9) is smaller than the number of ideal structures (13). Moreover, only six minima correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of beta-glucoside hydrolase Michaelis complexes. We show that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepared for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.

The enzymatic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymatic catalysis that extend beyond the bounds of carbohydrate chemistry, but the enzymes themselves play myriad essential roles in diverse biological processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiology and disease, has engendered a large scientific constituency with an interest in glycosidase chemistry. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational analysis of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme-ligand species adjacent to the transition state (or states) and of enzyme-inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mechanical basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative positive charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallographic observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the "electrophilic migration" of the anomeric center along the ...

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Removal of the catalytic nucleophile Glu134 of the retaining 1,3-1,4-L L-glucanase from Bacillus licheniformis by mutation to alanine yields an enzyme with no glycosidase activity. The mutant is able to catalyze the regio-and stereospecific glycosylation of K K-laminaribiosyl fluoride with different glucoside acceptors through a single-step inverting mechanism. The main advantage of the mutant as glycosylation catalyst with respect to the kinetically controlled transglycosylation using the wild-type enzyme is that the reaction products cannot be hydrolyzed by the mutant enzyme, and glycosylation yields rise to 90%.z 1998 Federation of European Biochemical Societies.

Cell signaling and other biological activities of chitooligosaccharides (COSs) seem to be dependent not only on the degree of polymerization, but markedly on the specific de-N-acetylation pattern. Chitin de-N-acetylases (CDAs) catalyze the hydrolysis of the acetamido group in GlcNAc residues of chitin, chitosan, and COS. A major challenge is to understand how CDAs specifically define the distribution of GlcNAc and GlcNH2 moieties in the oligomeric chain. We report the crystal structure of the Vibrio cholerae CDA in four relevant states of its catalytic cycle. The two enzyme complexes with chitobiose and chitotriose represent the first 3D structures of a CDA with its natural substrates in a productive mode for catalysis, thereby unraveling an induced-fit mechanism with a significant conformational change of a loop closing the active site. We propose that the deacetylation pattern exhibited by different CDAs is governed by critical loops that shape and differentially block accessible subsites in the binding cleft of CE4 enzymes.

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In familial amyloidotic polyneuropathy, TTR (transthyretin) variants are deposited as amyloid fibrils. It is thought that this process involves TTR tetramer dissociation, which leads to partially unfolded monomers that aggregate and polymerize into amyloid fibrils. This process can be counteracted by stabilization of the tetramer. Several small compounds, such as diclofenac, diflunisal and flufenamic acid, have been reported to bind to TTR in vitro, in the T4 (thyroxine) binding channel that runs through the TTR tetramer, and consequently are considered to stabilize TTR. However, if these agents bind plasma proteins other than TTR, decreased drug availability will occur, compromising their use as therapeutic agents for TTR amyloidosis. In the present work, we compared the action of these compounds and of new derivatives designed to increase both selectivity of binding to TTR and inhibitory potency in relation to TTR amyloid fibril formation. We found two diflunisal derivatives that, in contrast with diclofenac, flufenamic acid and diflunisal, displaced T4 from TTR in plasma preferentially over binding to albumin and thyroxine binding globulin. The same diflunisal derivatives also had a stabilizing effect on TTR tetramers in plasma, as studied by isoelectric focusing of whole plasma under semi-denaturing conditions. In addition, by transmission electron microscopy, we demonstrated that, in contrast with other proposed TTR stabilizers (namely diclofenac, flufenamic acid and diflunisal), one of the diflunisal derivatives tested efficiently inhibited TTR aggregation. Taken together, our ex vivo and in vitro studies present evidence for the selectivity and efficiency of novel diflunisal derivates as TTR stabilizers and as inhibitors of fibril formation.

Glycoside hydrolases that release fixed carbon from the plant cell wall are of considerable biological and industrial importance. These hydrolases contain noncatalytic carbohydrate binding modules (CBMs) that, by bringing the appended catalytic domain into intimate association with its insoluble substrate, greatly potentiate catalysis.