Glycosyltransferase-catalyzed Reactions Research Articles

Chemical Evolution of Enzyme-Catalyzed Glycosylation.

ConspectusThe limited availability of structurally well-defined diverse glycans remains a major obstacle for deciphering biological functions as well as biomedical applications of carbohydrates. Despite tremendous progress that has been made in past decades, the synthesis of structurally well-defined complex glycans still represents one of the most challenging topics in synthetic chemistry. Chemical synthesis of glycans is a time-consuming and labor-intensive process that requires elaborate planning and skilled personnel. In contrast, glycosyltransferase-catalyzed enzymatic synthesis provides a more efficient, convenient, low-cost, and sustainable alternative to affording diverse and complex glycans. However, the existing methods are still insufficient to fulfill the increasing demand for specific synthetic glycan libraries necessary for functional glycomics research. This is mainly attributed to the inherent character of the glycan biosynthetic pathway. In nature, there are too many glycosyltransferases involved in the in vivo glycan synthesis, but only a small number of them are available for in vitro enzymatic synthesis. For instance, humans have over 200 glycosyltransferases, but only a few of them could be produced from the conventional bacterial expression system, and most of these membrane-associated enzymes could be overexpressed only in eukaryotic cells. Moreover, the glycan biosynthetic pathway is a nontemplate-driven process, which eventually ends up with heterogeneous glycan product mixtures. Therefore, it is not a practical solution for the in vitro enzymatic synthesis of complex glycans by simply copying the glycan biosynthetic pathway.In the past decade, we have tried to develop a simplified and transformable approach to the enzymatic modular assembly of a human glycan library. Despite the structural complexity of human glycans, the glycoinformatic analysis based on the known glycan structure database and the human glycosyltransferase database indicates that there are approximately 56 disaccharide patterns present in the human glycome and only 16 disaccharide linkages are required to account for over 80% of the total disaccharide fragments, while 35 disaccharide linkages are sufficient to cover over 95% of all disaccharide fragments of human glycome. Regardless of the substrate specificity, if one glycosyltransferase could be used for the synthesis of all of the same glycosidic linkages in human glycome, it will require only a few dozen glycosyltransferases for the assembly of entire human glycans. According to the glycobioinformatics analysis results, we rationally designed about two dozen enzyme modules for the synthesis of over 20 common glycosidic linkages in human glycome, in which each enzyme module contains a glycosyltransferase and a group of enzymes for the in situ generation of a nucleotide-activated sugar donor. By sequential glycosylation using orchestrated enzyme modules, we have completed the synthesis of over 200 structurally well-defined complex human glycans including blood group antigens, O-mannosyl glycans, human milk oligosaccharides, and others. To overcome the product microheterogeneity problem of enzymatic synthesis in the nontemplate-driven glycan biosynthetic pathway, we developed several substrate engineering strategies to control or manipulate the outcome of glycosyltransferase-catalyzed reactions for the precise synthesis of structurally well-defined isomeric complex glycans.

Integrated Chemoenzymatic Approach to Streamline the Assembly of Complex Glycopeptides in the Liquid Phase

Glycosylation of proteins is a complicated post-translational modification. Despite the significant progress in glycoproteomics, accurate functions of glycoproteins are still ambiguous owing to the difficulty in obtaining homogeneous glycopeptides or glycoproteins. Here, we describe a streamlined chemoenzymatic method to prepare complex glycopeptides by integrating hydrophobic tag-supported chemical synthesis and enzymatic glycosylations. The hydrophobic tag is utilized to facilitate peptide chain elongation in the liquid phase and expeditious product separation. After removal of the tag, a series of glycans are installed on the peptides via efficient glycosyltransferase-catalyzed reactions. The general applicability and robustness of this approach are exemplified by efficient preparation of 16 well-defined SARS-CoV-2 O-glycopeptides, 4 complex MUC1 glycopeptides, and a 31-mer glycosylated glucagon-like peptide-1. Our developed approach will open up a new range of easy access to various complex glycopeptides of biological importance.

Biochemical characterization of Helicobacter pylori α1–3-fucosyltransferase and its application in the synthesis of fucosylated human milk oligosaccharides

Fucosylated human milk oligosaccharides (HMOs) have important biological functions. Enzymatic synthesis of such compounds requires robust fucosyltransferases. A C-terminal 66-amino acid truncated version of Helicobacter pylori α1–3-fucosyltransferase (Hp3FT) is a good candidate. Hp3FT was biochemically characterized to identify optimal conditions for enzymatic synthesis of fucosides. While N-acetyllactosamine (LacNAc) and lactose were both suitable acceptors, the former is preferred. At a low guanosine 5′-diphospho-β-L-fucose (GDP-Fuc) to acceptor ratio, Hp3FT selectively fucosylated LacNAc. Based on these enzymatic characteristics, diverse fucosylated HMOs, including 3-fucosyllactose (3-FL), lacto-N-fucopentaose (LNFP) III, lacto-N-neofucopentaose (LNnFP) V, lacto-N-neodifucohexaose (LNnDFH) II, difuco- and trifuco-para-lacto-N-neohexaose (DF-paraLNnH and TF-para-LNnH), were synthesized enzymatically by varying the ratio of the donor and acceptor as well as controlling the order of multiple glycosyltransferase-catalyzed reactions.

Strategies for chemoenzymatic synthesis of carbohydrates

Carbohydrates are structurally complex but functionally important biomolecules. Therefore, they have been challenging but attractive synthetic targets. While substantial progress has been made on advancing chemical glycosylation methods, incorporating enzymes into carbohydrate synthetic schemes has become increasingly practical as more carbohydrate biosynthetic and metabolic enzymes as well as their mutants with synthetic application are identified and expressed for preparative and large-scale synthesis. Chemoenzymatic strategies that integrate the flexibility of chemical derivatization with enzyme-catalyzed reactions have been extremely powerful. Briefly summarized here are our experiences on developing one-pot multienzyme (OPME) systems and representative chemoenzymatic strategies from others using glycosyltransferase-catalyzed reactions for synthesizing diverse structures of oligosaccharides, polysaccharides, and glycoconjugates. These strategies allow the synthesis of complex carbohydrates including those containing naturally occurring carbohydrate postglycosylational modifications (PGMs) and non-natural functional groups. By combining these srategies with facile purification schemes, synthetic access to the diverse space of carbohydrate structures can be automated and will not be limited to specialists.

Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

We described the integration of the general reversibility of glycosyltransferase-catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k(cat)/K(m) of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2-chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.

A Sialyltransferase Mutant with Decreased Donor Hydrolysis and Reduced Sialidase Activities for Directly Sialylating Lewisx

Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.

Using simple donors to drive the equilibria of glycosyltransferase-catalyzed reactions.

We report the ability of simple glycoside donors to drastically shift the equilibria of glycosyltransferase-catalyzed reactions, transforming NDP-sugar formation from an endo- to an exothermic process. To demonstrate the utility of this thermodynamic adaptability, we highlight the glycosyltransferase-catalyzed synthesis of 22 sugar nucleotides from simple aromatic sugar donors as well as the corresponding in situ formation of sugar nucleotides as a driving force in context of glycosyltransferase-catalyzed reactions for small molecule glycodiversification. These simple aromatic donors also enabled the first general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering, and other fundamental sugar nucleotide-dependent investigations. This study directly challenges the general notion that NDP-sugars are ‘high-energy’ sugar donors when taken out of their traditional biological context.

Glycosyltransferase Mechanisms: Impact of a 5-Fluoro Substituent in Acceptor and Donor Substrates on Catalysis

In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogues on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and beta-N-acetylglucosaminyl-beta-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc beta-octyl glycoside acceptor, however, is an excellent substrate for GalT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration.

Exploiting the Reversibility of Natural Product Glycosyltransferase-Catalyzed Reactions

Glycosyltransferases (GTs), an essential class of ubiquitous enzymes, are generally perceived as unidirectional catalysts. In contrast, we report that four glycosyltransferases from two distinct natural product biosynthetic pathways-calicheamicin and vancomycin-readily catalyze reversible reactions, allowing sugars and aglycons to be exchanged with ease. As proof of the broader applicability of these new reactions, more than 70 differentially glycosylated calicheamicin and vancomycin variants are reported. This study suggests the reversibility of GT-catalyzed reactions may be general and useful for generating exotic nucleotide sugars, establishing in vitro GT activity in complex systems, and enhancing natural product diversity.

Synthesis of unnatural sugar nucleotides and their evaluation as donor substrates in glycosyltransferase-catalyzed reactions

New unnatural sugar nucleotides, UDP-Fuc and CDP-Fuc were synthesized from fucose-β-1-phosphate and nucleotide monophosphates activated as morpholidates. Furthermore, a nucleotide analogue was prepared by phosphorylation of 1-(β- D-ribofuranosyl)cyanuric acid, itself obtained as a protected derivative by condensation of the persilylated derivative of cyanuric acid with 1- O-acetyl-2,3,5-tri- O-benzoyl-β- D-ribofuranose in 74% yield. This phosphate activated according to the same procedure was condensed with fucose-β-1-phosphate, affording a new sugar nucleotide conjugate (NDP-Fuc) which was evaluated together with UDP-Fuc, CDP-Fuc and ADP-Fuc, as fucose donors in α-(1→4/3)-fucosyltransferase (FucT-III) catalyzed reaction. Fucose transfer could be observed with each of the donors and kinetic parameters were determined using a fluorescent acceptor substrate. Efficiency of the four analogues towards FucT-III was in the following order: UDP-Fuc = ADP-Fuc > NDP-Fuc > CDP-Fuc. According to the same strategy ADP-GlcNAc was prepared from AMP-morpholidate and N-acetylglucosamine-α-1-phosphate; tested as a glucosaminyl donor towards Neisseria meningitidis N-acetylglucosaminyl transferase ( LgtA), ADP-GlcNAc was recognized with 0.1% efficiency as compared with UDP-GlcNAc, the natural donor substrate.

ChemInform Abstract: A Strategy for the Solution-Phase Parallel Synthesis of N-(Pyrrolidinylmethyl)hydroxamic Acids.

Both five- and six-membered iminocyclitols have proven to be useful transition-state analogue inhibitors of glycosidases. They also mimic the transition-state sugar moiety of the nucleoside phosphate sugar in glycosyltransferase-catalyzed reactions. Described here is the development of a general strategy toward the parallel synthesis of a five-membered iminocyclitol linked to a hydroxamic acid group designed to mimic the transition state of GDP-fucose complexed with Mn(II) in fucosyltransferase reactions. The iminocyclitol 8 containing a protected hydroxylamine unit was prepared from d-mannitol. The hydroxamic acid moiety was introduced via the reaction of 8 with various acid chlorides. The strategy is generally applicable to the construction of libraries for identification of glycosyltransferase inhibitors.

A strategy for the solution-phase parallel synthesis of N-(pyrrolidinylmethyl)hydroxamic acids.

Both five- and six-membered iminocyclitols have proven to be useful transition-state analogue inhibitors of glycosidases. They also mimic the transition-state sugar moiety of the nucleoside phosphate sugar in glycosyltransferase-catalyzed reactions. Described here is the development of a general strategy toward the parallel synthesis of a five-membered iminocyclitol linked to a hydroxamic acid group designed to mimic the transition state of GDP-fucose complexed with Mn(II) in fucosyltransferase reactions. The iminocyclitol 8 containing a protected hydroxylamine unit was prepared from D-mannitol. The hydroxamic acid moiety was introduced via the reaction of 8 with various acid chlorides. The strategy is generally applicable to the construction of libraries for identification of glycosyltransferase inhibitors.

Chemoenzymatic synthesis of sialyl-trimeric-Lewis x.

The decasaccharide sialyl-trimeric-Lewis x is a component of glycoproteins and glycolipids that serve as E- and P-selectin ligands. The synthesis of this target structure was accomplished by utilizing a combination of chemical and enzymatic methods. Highlights of the chemical synthesis include minimal use of protecting groups and regioselective glycosylations to arrive at a linear tri-lactosamine structure. Glycosyltransferase-catalyzed reactions were then employed for the addition of the terminal sialic acid and branch-point fucose residues. Notably, fucosyltransferases V and VI showed different specificities for the sialyl-tri-lactosamine core structure.

A Moenomycin-type Structural Analogue of Lipid II some possible mechanisms of the mode of action of transglycosylase inhibitors can be discarded

The transglycosylation step in the peptidoglycan biosynthesis belongs to the general class of glycosyltransferase-catalyzed reactions. The structural analogue 2 of moenomycin A has been synthesized and has been found to be antibiotically inactive. The assumption that moenomycin-type transglycosylase inhibitors such as 1 bind at the donor site of the enzyme and that their mode of action is the result of the high stability of the sugar phosphate bond seems to be ruled out by the present results.