Glycosynthesis in a waterworld: new insight into the molecular basis of transglycosylation in retaining glycoside hydrolases

Abstract

Carbohydrates are ubiquitous in Nature and play vital roles in many biological systems. Therefore the synthesis of carbohydrate-based compounds is of considerable interest for both research and commercial purposes. However, carbohydrates are challenging, due to the large number of sugar subunits and the multiple ways in which these can be linked together. Therefore, to tackle the challenge of glycosynthesis, chemists are increasingly turning their attention towards enzymes, which are exquisitely adapted to the intricacy of these biomolecules. In Nature, glycosidic linkages are mainly synthesized by Leloir glycosyltransferases, but can result from the action of non-Leloir transglycosylases or phosphorylases. Advantageously for chemists, non-Leloir transglycosylases are glycoside hydrolases, enzymes that are readily available and exhibit a wide range of substrate specificities. Nevertheless, non-Leloir transglycosylases are unusual glycoside hydrolases in as much that they efficiently catalyse the formation of glycosidic bonds, whereas most glycoside hydrolases favour the mechanistically related hydrolysis reaction. Unfortunately, because non-Leloir transglycosylases are almost indistinguishable from their hydrolytic counterparts, it is unclear how these enzymes overcome the ubiquity of water, thus avoiding the hydrolytic reaction. Without this knowledge, it is impossible to rationally design non-Leloir transglycosylases using the vast diversity of glycoside hydrolases as protein templates. In this critical review, a careful analysis of literature data describing non-Leloir transglycosylases and their relationship to glycoside hydrolase counterparts is used to clarify the state of the art knowledge and to establish a new rational basis for the engineering of glycoside hydrolases.

When substrate inhibits and inhibitor activates: implications of β-glucosidases

“…Besides hydrolysis, many BGs have been shown to catalyze also transglycosylation reactions [8–10, 19, 60]. First, we consider the transglycosylation reactions involving inhibitors.…”

“…GHs within these families employ the retaining catalytic mechanism that involves a covalent glycosyl-enzyme intermediate. Therefore, the direct reverse reaction, i.e., between two product molecules, is unlikely in aqueous environment [60, 61]. The double displacement mechanism of retaining GHs can be divided into two steps.…”

Comparison of lipases and glycoside hydrolases as catalysts in synthesis reactions

“…Some enzymes have strong homology with normal glycoside hydrolases but catalyze mainly transglycosylation reactions (Bissaro et al 2015). One example is the glucansucrases, classified in glycoside hydrolase family 70.…”

Glycosyltransferase engineering for carbohydrate synthesis

“…Several other assays for GT activity measure nucleotide sugar depletion or nucleotide diphosphate formation [3–7]. These types of assays are appealing because they are universal for all GT reactions that use nucleotide diphosphate-sugars.…”

“…Many advances have been made in chemical glycosylation [1–6]. With an improved understanding of their catalytic mechanisms, enzyme-catalyzed processes using glycosidases, trans-glycosidases, and glycosidase-derived glycosynthases have been developed successfully [7–9]. Glycosyltransferases (GTs), the key enzymes used by nature in producing diverse carbohydrate-containing structures, continue to be important catalysts for accessing complex oligosaccharides and glycoconjugates as well as for glycan modification of glycoconjugates and cell surface glycans [10, 11].…”