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.

Keywords: Leloir; applied biocatalysis; carbohydrate; chemoenzymatic synthesis; enzyme cascades; glycosyltransferase; nucleotide; sugar chemistry.

Figure 1

The nomenclature of glycosides and oligosaccharides.

Scheme 1

The overall scheme of an enzymatic glycosylation reaction for the biocatalytic synthesis of glycosides by retaining or inverting glycosyltransferases (GT) using NDP or P_i activated sugar donors for Leloir and non-Leloir GTs, respectively.

Figure 2

Common sugar nucleotides found in all kingdoms of life. Abbreviations: UDP-Glc, UDP-glucose; UDP-GalNAc, UDP-N-acetyl-2-deoxy-d-galactosamine; UDP-GalA, UDP-d-galacturonic acid; UDP-GlcA, UDP-d-glucuronic acid; UDP-GlcNAc, UDP-N-acetyl-2-deoxy-d-glucosamine; UDP-FucNAc, UDP-N-acetyl-l-fucosamine; UDP-Gal, UDP-d-galactose; CMP-Kdo, CMP-3-deoxy-d-manno-octulosonate; CMP-Sia, CMP-N-acetylneuraminic acid; GDP-Fuc, GDP-l-fucose; GDP-Man, GDP-d-mannose; ADP-HEP, ADP-l-glycero-d-manno-heptose.

Figure 3

Protein folds of Leloir glycosyltransferases (GT-A, GT-B, GT-D) and non-Leloir glycosyltransferases (GT-C, GT-E).

Figure 4

Reaction mechanism of glycosyltransferases upon inversion (a) or retention (b,c) of the anomeric glycosidic bond. The divalent metal (M²⁺) is not necessarily a requirement for catalytic activity for GTs.

Figure 5

Exemplary enzymatic glycosylation of an activated sugar donor (green) and acceptor (R-group) to afford a maximum transient kinetic (blue) product yield catalyzed by a glycoside hydrolase, followed by reverse hydrolysis towards the thermodynamic product concentration. Direct esterification leads to the thermodynamic product yield K_eq without the requirement for an activated sugar (red) in (a). LeLoir GTs only catalyze the direct esterification of a nucleotide sugar donor (purple) to thermodynamic product (red) in (b).

Figure 6

Glycosylation of aglycones producing phenolic glycosides, amino glycosides, alcohol glycosides, ester glycosides, and disaccharides with their estimated K_eq. The K_eq was calculated from the Gibbs free energy ΔG_r′° using the eQuilibrator web interface (http://equilibrator.weizmann.ac.il) [155] assuming the following conditions: ionic strength 0.1 M, pH 7.0, aglycon (1 mM), UDP (1 mM), UDP-d-glucose (1 mM), glycosylated product (1 mM), and 298 K.

Figure 7

Enzymatic cascade for the production of stachyose from sucrose with glycosyltransferases. (a). The standard Gibbs free energy changes of the individual reactions (ΔG°s, red) and the total reaction (ΔG°, grey) shown in (b) [183]. The Δ_rG′° represents the change of Gibbs free energy and was calculated using the eQuilibrator web interface (http://equilibrator.weizmann.ac.il) [155] using the following conditions: ionic strength 0.1 M, pH 7.0, 1 mM of component, 298 K. Abbreviations: UDP-d-glc, UDP-d-glucose; UDP-d-gal, UDP-d-galactose, SuSy, Sucrose synthase; GalE, UDP-d-glucose-4-epimerase; GS, galactinol synthase; RS, raffinose synthase; STS, stachyose synthase.

Figure 8

The use of different energy-rich phosphate donors to regenerate NTP using either pyruvate- (a), creatine- (b), or acetate kinase (c). The Δ_rG′° represents the standard change of Gibbs free energy and was calculated using the eQuilibrator web interface (http://equilibrator.weizmann.ac.il) [155] using the following conditions: ionic strength 0.1 M, pH 7.0, 1 mM of component, 298 K. Abbreviations: NDP, nucleotide diphosphate; NTP, nucleotide triphosphate; ADP, adenosine diphosphate; UDP, uridine diphosphate; CDP, cytidine diphosphate; dTDP; deoxythymidine diphosphate; PEP, phosphoenolpyruvate.

Figure 9

Several sacrificial phosphate donors for sugar nucleotide (re)generation systems of galactosyltransferases using a stoichiometric amount of NTPs (a) [199], PEP (b) [200], PolyP_n (c) [189], and acyl P_i (d) [201]. Abbreviations: PP_i, pyrophosphate; P_i, orthophosphate; UMP, uridine monophosphate; UDP, uridine diphosphate; UTP, uridine triphosphate; UDP-Gal, UDP-d-galactose; ADP, adenosine diphosphate; ATP, adenosine triphosphate; Gal, d-galactose; Gal1P, d-galactose-1-phosphate; PEP, (phospho)enol pyruvate; PolyP_n, (poly)phosphate; acyl P_i, acetyl phosphate.

Figure 10

Enzymatic glycosylation for the production of N-acetyl-d-lactosamine from glucose-6-phosphate and N-acetyl-d-glucosamine (a) [208]. The Δ_rG′° represents the standard change in Gibbs free energy (b) was calculated using the eQuilibrator web interface (http://equilibrator.weizmann.ac.il) [155] using the following conditions: ionic strength 0.1 M, pH 7.0, 1 mM of component, 298 K. Abbreviations: Glc-6-P, d-glucose-6-phosphate; Glc-1-P, d-glucose-1-phosphate; P₂O₇⁴⁻, pyrophosphate; HPO₄²⁻, orthophosphate; UDP, uridine diphosphate; PGM, Phosphoglucomutase; UDPG-P, UDP-glucose pyrophosphorylase; GalE, UDP-galactose epimerase; GalT, galactosyltransferase; PK, pyruvate kinase.

Figure 11

Reaction scheme of both the multistep chemical glycosylation utilizing previously synthesized carbohydrate building blocks [221] and enzymatic glycosylation utilizing nucleotide sugar donors for linear saccharide elongation capped with a terminal vinyl group [129]. The top half of the figure was adapted from [221], copyright 2006, National Academy of Sciences.

Figure 12

The enzymatic synthesis of GD1b glycan using OPME α2,3-sialylation, α3,8-sialylation, β1,4-N-acyl-galactosylation, and β1,3-galactosylation with a sacrificial (re)generation system for N-acetylneuraminic acid (Neu5Ac), N-acetylgalactosamine (GalNAc), and d-galactose (Gal).

Figure 13

Examples of oligosaccharides synthesized with automated enzymatic synthesis where GM1 is a well-known ganglioside, and the antigens of blood types A, B, and O.

Figure 14

Different modes of immobilization of Leloir glycosyltransferases with entrapment, cross-linking, or carrier-bound attachment.

Figure 15

Different reactor types for the immobilization of Leloir glycosyltransferases.

Figure 16

Glass microchannel reactor with immobilized enzyme sucrose phosphorylase attached to the siliceous wall [277]. Attachment occurs via the highly positively charged Z_basic2 binding module, which binds to the negatively charged silica surface.