Leloir types

Highly selective enzymatic synthesis using glycosyltransferases (GTFs), an approach restricted by the limited availability of Leloir-type enzymes, and expensive nucleotide-activated substrates... 

Sucrase-type (non-Leloir-type) enzymes that operate both regio- and stereo-selectively, using sucrose as a cheap substrate, or, in some cases (such as cyclodextrin (CD) transferases) starch these enzymes are, however, limited to the transfer of only glucose or fructose... 

Scheme 2.2.5.25 Enzymatic activation of sialic acids to Leloir-type sugar nucleotides using the CMP-NeuAc synthetase of Neisseria meningitidis.

The first member of the GT-A superfamily fold was identified in 1999 when the three-dimensional stmcture of SpsA was reported (19). This enzyme from Bacillus subtilis is involved in the formation of the spore coat and is a member of the Leloir type of GTs given its apparent use of nucleotidyl-diphospho-donor sugars as a substrate. Enzymes with the GT-A fold have an N-terminal a/p/a sandwich motif that resembles a Rossmann motif and is involved in nucleotide donor binding. Most GT-A fold enzymes also have a characteristic Asp-Xxx-Asp (DXD) or equivalent motif (EXD or TDD) near the center of the protein that coordinates to the phosphate in nucleotide donors via a divalent metal cation (Mn + or Mg +). These motifs are present in the structure of the bovine pi,4-galactosyltransferase I (5) (Fig. 4a). 

It follows from the above that, if highly specific oligosaccharides need to be synthesized, glycosyltransferases [21] of the Leloir type [22] should be the enzymes of choice. These enzymes connect sugars via the activated nucleotide components with high stereo- and regioselectivity. They are substrate-specific, but, in vitro, with solubilized enzymes, it becomes possible to transfer modified donors to modified acceptors and thus broaden the scope of the synthetic applicability. 

In this context, the use of enzymes has emerged as a practical alternative to chemical synthesis [6-13]. Several examples have been reported that are based on the use of Leloir type-glycosyltransferases, which are mostly membrane-associated and act on nucleotide-activated sugars as donor substrates. However, the use of these enzymes in vitro still remains limited by the difficulty of enzyme purification and by their need for expensive sugar-nucleotides [6]. Re-engineering of microbial cells producing these proteins appears to be promising for the synthesis of specific carbohydrate stmctures [14]. 

Leloir-type glycosyltransferases are typically membrane bound. In this regard, the use of immobilized enzyme systems [38] may advantageously create enhanced stability one ready method is the use of affinity supports [39, 40]. In addition, the membrane bound nature of Gly-Ts may require the removal of an enzyme s membrane-binding domain to ensure solubility. 

Leaving Group (LG) = nucleotide(di)phosphate for Leloir type Figure 12.14... 

This chapter will describe several Leloir-type transferase reactions that have been used for stepwise construction of a oligosaccharides comprising five different monosaccharides, GlcNAc, GalNAc, Gal, Fuc, and Neu5Ac. This is a very limited approach among the myriads of possible oligosaccharides that can be potentially... 

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