Michael Jahn

Expansion of the glycosynthase repertoire to produce defined manno-oligosaccharides

Mutant endo-mannanases, in which the catalytic nucleophile has been replaced, function as glycosynthases in the synthesis of manno-oligosaccharides of defined lengths.

Thioglycosynthases: double mutant glycosidases that serve as scaffolds for thioglycoside synthesis

A double mutant, retaining glycosidase that lacks both the catalytic nucleophile and the catalytic acid/base residues efficiently catalyzes thioglycoside formation from a glycosyl fluoride donor and thiosugar acceptors.

Thermostable Glycosynthases and Thioglycoligases Derived from Thermotoga maritima β-Glucuronidase

Uronic acid-containing glycoconjugates are found in a number of different and important contexts. Examples include pectins and hemicelluloses within plant cell walls, glycosaminoglycans (GAGs) within the mammalian extracellular matrix, capsular poly ACHTUNGTRENNUNGsaccharides of bacteria and glucuronide conjugates formed as a means of solubilisation and clearance of unwanted molecules. Of these, GAGs such as heparin, heparan sulfate, chondroitin sulfate and hyaluronan are of particular importance in a variety of biological processes, and analogues of these structures might well be useful as therapeutic agents. 2] While the chemical syntheses of short oligosaccharide fragments of GAGs have been achieved, these syntheses are surprisingly challenging, and their scale-up to the levels needed for clinical trials remains challenging. An alternative synthetic approach involves the use of enzymes, and, in that regard, ACHTUNGTRENNUNGadvances have been made in several areas. The enzymes ACHTUNGTRENNUNGinvolved in GAG biosynthesis, the glycosyltransferases, have proved problematic, largely because they are generally closely membrane-associated. However, considerable success has been achieved by DeAngelis’ group with hyaluronan synthase, and small-scale syntheses with recombinant enzymes are now available. 5] Glycosidases run “in reverse” provide the other approach, and, indeed, Kobayashi et al. have successfully assembled hyaluronan and chondroitin sulfate oligosaccharides from disaccharide precursors, converted into their oxazolines, by use of endo-hexosaminidases. 7] An alternative strategy for oligosaccharide assembly involves the use of retaining glycosidases in which either the catalytic nucleophile (glycosynthases) or the acid/base catalyst (thioACHTUNGTRENNUNGglycoligases) is mutated (Scheme 1). Glycosynthases are hydroACHTUNGTRENNUNGlytically incompetent mutants that can, nevertheless, effect efficient glycosyl transfer from a glycosyl fluoride sugar donor of opposite anomeric configuration to that of the natural substrate. The glycosyl fluoride donor binds and acts as a mimic of the normal glycosyl enzyme. A range of such enzymes has now been produced, and directed evolution has generated highly efficient catalysts (kcat 90 s ). Thioglycoligases carry out glycosyl transfer from an activated glycosyl donor of normal configuration to a thiosugar acceptor, with formation of a thioglycosidic linkage. In this case, the activated leaving group “complements” the absence of the acid catalyst in the formation of the glycosyl enzyme, while the highly nucleophilic thiol ACHTUNGTRENNUNG(ate) “complements” the missing general base catalyst. While a range of glycosynthases and thioglycoligases has now been produced, and directed evolution approaches have been employed to boost rates and alter specificities, there have been no reports to date of either glycosynthases or thioglycoligases that transfer glycuronyl residues. Given the importance of these structures and the difficulties noted earlier with effective synthesis, we investigated the potential for both classes of mutant enzymes in the synthesis of glycuronyl linkages. An appropriate candidate glycuronidase for the generation of both a glycosynthase and a thioglycoligase was the thermostable b-glucuronidase of Thermotoga maritima (TMGUA), a member of GH family 2 (http://afmb.cnrs-mrs.fr/ CAZY/), particularly as the identities of both the nucleophile (E476) and the acid/base catalyst (E383) have recently been confirmed.

New Approaches to Enzymatic Oligosaccharide Synthesis: Glycosynthases and Thioglycoligases

Abstract An overview of the applications of engineered glycosynthases and thioglycoligases for the enzymatic synthesis of O- and S-glycosidic linkages in oligosaccharides is presented. Glycosynthases lack the catalytic nucleophile of retaining glycosidases and use glycosyl fluorides with inverted anomeric stereochemistry as glycosyl donors. To date, nine enzymes from seven different glycosyl hydrolase families have been engineered to perform the glycosynthase reaction. Thioglycoligases lack the catalytic acid/base residue of retaining glycosidases and use dinitrophenyl glycosides as donors and deoxy-thiosugars as acceptors. The regioselectivity of the transglycosylation reaction is entirely controlled by the position of the thiol in the acceptor. To date, two retaining exo glycosidases and one endo glycanase, all from different glycosyl hydrolase families, have been engineered in this fashion.

Activity of three β-1,4-galactanases on small chromogenic substrates.

β-1,4-Galactanases belong to glycoside hydrolase family GH 53 and degrade galactan and arabinogalactan side chains of the complex pectin network in plant cell walls. Two fungal β-1,4-galactanases from Aspergillus aculeatus, Meripileus giganteus and one bacterial enzyme from Bacillus licheniformis have been kinetically characterized using the chromogenic substrate analog 4-nitrophenyl β-1,4-d-thiogalactobioside synthesized by the thioglycoligase approach. Values of k(cat)/K(m) for this substrate with A. aculeatus β-1,4-galactanase at pH 4.4 and for M. giganteus β-1,4-galactanase at pH 5.5 are 333M(-1)s(-1) and 62M(-1)s(-1), respectively. By contrast the B. licheniformis β-1,4-galactanase did not hydrolyze 4-nitrophenyl β-1,4-d-thiogalactobioside. The different kinetic behavior observed between the two fungal and the bacterial β-1,4-galactanases can be ascribed to an especially long loop 8 observed only in the structure of B. licheniformis β-1,4-galactanase. This loop contains substrate binding subsites -3 and -4, which presumably cause B. licheniformis β-1,4-galactanase to bind 4-nitrophenyl -1,4-β-d-thiogalactobioside non-productively. In addition to their cleavage of 4-nitrophenyl -1,4-β-d-thiogalactobioside, the two fungal enzymes also cleaved the commercially available 2-nitrophenyl-1,4-β-d-galactopyranoside, but kinetic parameters could not be determined because of transglycosylation at substrate concentrations above 4mM.