Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach

Abstract

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio-and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.hydroxynitrile lyases catalyzed the enzymatic hydrolysis of the glycoside amygdalin [3]. Moving almost two centuries forward, the largest volumetric biocatalytic industrial process is the application of glucose isomerase for the production of high fructose syrup for food and drink applications, producing fructose from glucose at 10 7 tons per year [4]. The secret of the success of enzymes in the production or treatment of carbohydrates and glycosides is their exquisite stereo-and regioselectivity. The excellent selectivity of enzymes is required due to the diversity of structural features of carbohydrates [5], comprising d-and l-epimers, ring size, anomeric configuration, linkages, branching, and oxidation state(s). Since drug targets often exhibit specificity for all of these structural features, the production process should not contain any side-products to prevent undesired side-effects [6].The challenge in the synthesis of carbohydrates is their wide variety of functionalities and stereochemistry ( Figure 1). (Poly)hydroxyaldehydes containing a terminal aldehyde are referred to as aldoses and (poly)hydroxyketones are defined as ketoses. In aqueous solutions, monosaccharides form equilibrium mixtures of linear open-chain and ring-closed 5-or 6-membered furanoses or pyranoses, respectively. For aldoses, the asymmetric ring forms at C-1. For ketoses, it closes at C-2 as an axial (α) or equatorial (β) hemiacetal or hemiketal, respectively (commonly defined as the anomeric center). A glycosidic linkage is a covalent O-, S-, N-, or C-bond connecting a monosaccharide to another residue resulting in a glycoside, while glucoside is specific for a glucose moiety. The equatorial or axial position of the glycosidic bond is referred to as α-(axial) or β-linkage (equatorial). The number of carbohydrates linked via glycosidic bonds can be subdivided into oligosaccharides with two to ten linked carbohydrates, while polysaccharides (glycans) contain more than ten glycosidic bonds. A glycan either con...

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

“…Glycosyltransferases (GTs) catalyze the formation of glycoside bonds using an activated donor sugar substrate that contains a nucleoside phosphate or a lipid phosphate leaving group [29]. Enzymes that use sugar mono-or diphosphonucleotides as donors are known as Leloir GTs [30]. Non-Leloir GTs use non-nucleotide donors, such as polyprenol pyrophosphates, polyprenol phosphates, sugar-1-phosphates, or sugar-1-pyrophosphates.…”

Comparative Analysis of High-Throughput Assays of Family-1 Plant Glycosyltransferases

“…GT family-1 of the CAZy classification, also referred to as UDP GTs (UGTs), are the largest GT family in plants that catalyze the transfer of a glycosyl moiety from UDP sugars to a wide range of small molecule acceptors [13]. Crystal structures from various plant UGTs have been analyzed and despite relatively low sequence identities, they all possess the GT-B fold consisting of two Rossmann domains -β/α/β [2,11,[15][16][17][18]. The majority of GT-A fold enzymes require divalent cations such as Mg 2+ and Mn 2+ for enzymatic activity and contain a DxD motif involved in the binding of the metal.…”

Immobilization of the Highly Active UDP-Glucose Pyrophosphorylase From Thermocrispum agreste Provides a Highly Efficient Biocatalyst for the Production of UDP-Glucose

“…It is one of the key precursors for sugar interconversion, for formation of di- and polysaccharides, and in amino and nucleotide sugar metabolism. In addition, UDP-glucose can be used as a source for other industrial interesting compounds such as antibiotics ( Lai et al, 2008 ; Rodríguez-Díaz and Yebra, 2011 ; Gutmann and Nidetzky, 2016 ; Huang et al, 2016 ; Schmölzer et al, 2017 ; Mestrom et al, 2019 ; Liu et al, 2020 ). A number of chemical methods for UDP-glucose synthesis have already been proposed ( Moffatt and Khorana, 1958 ; Hanessian et al, 1998 ), but they create reactivity and selectivity problems, often requiring modification of functional groups to protect those residues of the sugar molecules that should not react, and at the same time expose those groups that should react ( Guo and Ye, 2010 ).…”