@article { ISI:000166952700005,
	title = {Mechanisms of glycosyl transferases and hydrolases},
	journal = {CARBOHYDRATE POLYMERS},
	volume = {44},
	number = {4},
	year = {2001},
	note = {Eurocarb Conference, GALWAY, IRELAND, JUL 11-16, 1999},
	month = {APR},
	pages = {325-337},
	publisher = {Eurocarb},
	abstract = {Glycosidases and glycosyl transferases fall into two major mechanistic classes; those that hydrolyse the glycosidic bond with retention of anomeric configuration and those that do so with inversion. There are, however, two classes of transferases: those that use nucleotide phosphosugars (NP-sugar-dependent) and those that simply transglycosylate between oligosaccharides or polysaccharides (transglycosylases). The latter are mechanistically similar to retaining glycosidases while the mechanisms of NP-sugar-dependent transferases are far from clear. Retaining glycosidases and the transglycosylases employ a mechanism involving a covalent glycosyl-enzyme intermediate formed and hydrolysed with acid/base catalytic assistance via oxocarbenium ion-like transition states. This intermediate has been trapped on glycosidases in two distinct ways, either by modification of the substrate through fluorination, or of the enzyme through mutation of key residues. A third method has been developed for trapping the intermediate on transglycosylases involving the use of incompetent substrates that allow formation of the intermediate, but prohibit its transfer as a consequence of their acceptor hydroxyl group being removed. Three-dimensional structures of several of these glycosyl-enzyme complexes, along with those of Michaelis complexes, have been determined through X-ray crystallographic analysis, revealing the identities of important amino acid residues involved in catalysis. In particular they reveal the involvement of the carbonyl oxygen of the catalytic nucleophile in strong hydrogen bonding to the sugar 2-hydroxyl for the beta -retainers or in interactions with the ring oxygen for alpha -retainers. The glucose ring in the -1 (cleavage) site in the intermediates formed on several cellulases and a beta -glucosidase adopts a normal C-4(1) chair conformation. By contrast the xylose ring at this site in a xylanase is substantially distorted into a B-2,B-5 boat conformation, an observation that bears significant stereoelectronic implications. Substantial distortion is also observed in the substrate upon binding to several beta -glycosidases, this time to a S-1(3) skew boat conformation. Much less distortion is seen in the substrate bound on an alpha -transglycosylase. Finally an efficient catalyst for synthesis, but not hydrolysis, of glycosidic bonds has been generated by mutation of the glutamic acid catalytic nucleophile of a beta -glucosidase to an alanine. When used with alpha -glucosyl fluoride as a glycosyl donor, along with a suitable acceptor, oligosaccharides up to five sugars in length have been made with yields of up to 90\% on individual steps. These new enzymes have been named Glycosynthases. (C) 2001 Elsevier Science Ltd. All rights reserved.},
	issn = {0144-8617},
	doi = {10.1016/S0144-8617(00)00249-6},
	author = {Withers, SG}
}
