Donor Substrate Regeneration for Efficient Synthesis of Globotetraose and Isoglobotetraose

Abstract

Here we describe the efficient synthesis of two oligosaccharide moieties of human glycosphingolipids, globotetraose (GalNAc␤133Gal␣134Gal␤134Glc) and isoglobotetraose (GalNAc␤133Gal␣133Gal␤13 4Glc), with in situ enzymatic regeneration of UDP-N-acetylgalactosamine (UDP-GalNAc). We demonstrate that the recombinant ␤-1,3-N-acetylgalactosaminyltransferase from Haemophilus influenzae strain Rd can transfer N-acetylgalactosamine to a wide range of acceptor substrates with a terminal galactose residue. The donor substrate UDP-GalNAc can be regenerated by a six-enzyme reaction cycle consisting of phosphoglucosamine mutase, UDP-N-acetylglucosamine pyrophosphorylase, phosphate acetyltransferase, pyruvate kinase, and inorganic pyrophosphatase from Escherichia coli, as well as UDP-N-acetylglucosamine C4 epimerase from Plesiomonas shigelloides. All these enzymes were overexpressed in E. coli with six-histidine tags and were purified by one-step nickel-nitrilotriacetic acid affinity chromatography. Multiple-enzyme synthesis of globotetraose or isoglobotetraose with the purified enzymes was achieved with relatively high yields.

Improving N‐glycosylation efficiency in Escherichia coli using shotgun proteomics, metabolic network analysis, and selective reaction monitoring

“…Engineering E. coli to synthesize glycan groups such as N-acetylgalactoseamine (GalNAc), rather than providing an exogenous source is useful mainly because the free hydroxyl group on GalNAc reduces the membrane transport efficiency (Sarkar et al, 1995). In which case, enzymatic regeneration of the sugar nucleotide donor UDP-GalNAc, which is transferred onto the polypeptide backbone via C. jejuni glycosyltransferases, has been previously achieved using a six-enzyme pathway (Shao et al, 2002). However, this method is subject to a relatively high cost of UDP-GalNAc (approximately $1,000 per 100 mg), a commercial disadvantage of using this in vitro approach.…”

“…First, nucleotide sugar metabolism (left auxiliary pathway) contains 4 UDP-GalNAc regeneration pathway enzymes (Shao et al, 2002). In the iTRAQ discovery stage, the abundance of two of these enzymes were monitored (phosphoglucosamine mutase and L-glutamine:D-fructose-6-phosphate aminotransferase).…”

Efficient Enzymatic Glycosylation of Peptides and Oligosaccharides from GalNAc and UTP

“…[16] More recently, engineered bacteria or yeasts were developed which allow large scale synthesis of oligosaccharides with the recycling of some of the necessary sugar nucleotides. [22][23][24][25] GalNAc-containing glycoconjugates are among the most thoroughly studied blood group, oncofetal, or bacterial antigens and are in high demand for in vivo studies. In the method described by Shao et al [22] UDP-GalNAc regeneration was achieved in a bacterial system employing six enzymes including an epimerase to convert UDP-GlcNAc into UDP-GalNAc.…”

Approaches to the Enzymatic Carbohydrate Synthesis

“…Sialyltransferases are unique in that the glycosyl donor is activated by cytidine monophosphate (CMP-Neu5Ac). Recent advances in the area of enzymatic oligosaccharide synthesis are driven by the identification and cloning of a large number of bacterial glycosyltransferases with many different donor, acceptor and linkage specificities [18][19][20][21][22]. Most eukaryotic glycosyltransferases are not active within prokaryotic expression systems because of the absence of post-translation modifications including glycosylation.…”