Nucleotides sugar and

A rather limited collection of simple precursor molecules is sufficient to provide for the biosynthesis of virtually any cellular constituent, be it protein, nucleic acid, lipid, or polysaccharide. All of these substances are constructed from appropriate building blocks via the pathways of anabolism. In turn, the building blocks (amino acids, nucleotides, sugars, and fatty acids) can be generated from metabolites in the cell. For example, amino acids can be formed by amination of the corresponding a-keto acid carbon skeletons, and pyruvate can be converted to hexoses for polysaccharide biosynthesis. 

Appropriate nucleotide sugars and highly specific Golgi-located glycosyltransferases are employed to synthesize the oligosaccharide chains of GAGs. The one enzyme, one linkage relationship appears to hold here,... 

On the other hand, the enzymatic synthesis of glycoconjugates and oligosaccharides leads to high product yields in a short time by stereo- and regioselective one-step reactions. All enzymatic reactions are easy to scale up and are carried out in aqueous media under mild conditions. A whole set of enzymes is now available to build up OAT bonds in monosaccharides, COP bonds in activated monosaccharides e.g. phosphorylated sugars or nucleotide sugars, and C-O-C bonds in di- and oligosaccharides (Fig. 1). However, all these enzymatic reactions are limited by the substrate spectrum of the individual enzyme. 

In this context, the aim of our work is to provide well defined enzyme systems, to utilize them for the synthesis of these partly instable and very expensive nucleotide sugars, and finally to combine them with glycosyltransferases for glycoconjugate synthesis. In the following sections, I will present a summary of our work on the basis of the biosynthesis pathways for primary and secondary nucleotide sugars. 

The synthesis of monomer building blocks of cell-wall polymers, such as nucleotide sugars and monolignols... 

Gibeaut, D.M., 2000, Nucleotide sugars and glycosyltransferases for synthesis of cell wall matrix polysaccharides. Plant Physiol. Biochem. 38 69-80. 

Enzymes underlie our ability to move, to think, to sense our world. Enzymes help us wink an eye, savor an ice cream cone, and catch a sticky drip about to fall off the edge of the cone. Enzymes, and their essential cellular associates—other proteins, nucleotides, sugars, and fats—allow a stubbed toe... 

New Drugs by Expression of Artificial Genes UrdGTlb and UrdGTlc, involved in urdamycin biosynthesis, share 91% identical amino acids. However, the two GTs show different specificities for both nucleotide sugar and acceptor substrate. Targeted amino acid exchanges reduced the number of amino acids, potentially... 

Elling L, Grothus M, Kula MR. Investigation of sucrose synthase from rice for the synthesis of various nucleotide sugars and saccharides. Glycobiology 1993 3 349-355. 

Sequential biocatalytic cascade reactions are characterized by the use of multiple enzymatic steps involving various biocatalysts. One cascade reaction can consist of an enzyme-module with several enzymes if substrate and inhibitor kinetics are compatible with these combinations. Sequential use of such enzyme-modules surpassing the work-up of intermediate products is the criterion for the idea of cascade reactions we address here on the one hand, the synthesis of nucleotide sugars and their derivatives, on the other hand, the synthesis of glycan epitopes with multiple GTs. 

Enzymatic one-pot syntheses for the production of glycoconjugates are characterized by the use of multiple enzymes in one reaction vessel, therefore comprising multiple catalytic steps in situ. In comparison to sequential syntheses, the one-pot approach is the most favorable to optimize reaction time and space-time yield. However, as multiple enzymes are combined directly, the conditions must be carefuUy evaluated to suit all necessities of the involved biocatalysts. As in the previous part of this chapter, we take a look at some examples for the syntheses of nucleotide sugars and glycan structures. 

CMP-N-acetylneuraminic acid is formed by a series of reactions which are outgrowths of the pathways of hexosamine biosynthesis. As seen from Fig. 8A, N-acetylmannosamine can be regarded as the first specific intermediate in the biosynthesis of the nucleotide sugar and may be formed by 2-epimerization from either UDP-N-acetylglucosamine or N-acetylglucosamine. The mechanisms of these two reactions have recently been investigated and will be discussed below. 

Fig. 9. The reaction catalyzed by the sialytransferases acyl is either acetyl or glycolyl. The assay method for these transferases usually involves incubation of acceptor, enzyme, and radioactive CMP-slallc acid followed by high voltage paper electrophoresis in 1% sodium tetraborate (Roseman et

The requirements for the rat and pork liver sialyltransferases are shown in Table VI. The need for exogenous acceptor is almost absolute and neuraminidase-treated i-acid glycoprotein is far more eflFective than other derivatives, indicating that a terminal galactose residue is required for acceptor activity. The sialyltransferases show no requirements for cations, and the pH optima are 5.7 and 7.0 for the rat and pork liver enzymes, respectively. The Km values for the pork liver enzyme are 0.19 mM for the nucleotide sugar and 0.9 mM for the glycoprotein acceptor (calculated on the basis of available acceptor sites). 

UDP-Glucuronate carboxylyase is quite specific for the glycosyl moiety of the nucleotide sugar and does not, e.g., decarboxylate UDP-galacturonic acid. However, TDP-ghicuronate is a substrate for the enzyme, although the reaction velocity is only 11% of that observed with UDP-glucuronate under identical reaction conditions. 

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