Abstract
Abstract Carbohydrates and their glycoconjugates are important in mediating structural and functional roles in numerous physiological processes, including various disease states. Despite significant advancement in the field like programmable one-pot assembly of carbohydrates, at the recent time, synthesis of complex carbohydrates and glycoconjugates remains elusive than that of other biomolecules. To simplify the synthesis of carbohydrates, enzymes provide an alternative means that are likely to be synthetically viable to chemists. In this regard, enzymes like glycosyltransferases and glycosidases have proven useful biocatalysts in constructing stereo- and regiospecific glycosidic linkages in complex carbohydrate structures. However, in the preparative-scale synthesis point, glycosyltransferases from microbial sources may exhibit greater flexibility because of their ability to synthesize a large range of oligosaccharide analogues at relatively high yields. My dissertation describes the expression of various recombinant bacterial enzymes; thymidylyltransferase (RmlA) of Aneurinibacillus thermoaerophilus, N-acetylhexosamine-1-kinase (NahK) of Bifidobacterium longum, β-1,3-N-acetyl-glucosaminyltransferase of Helicobacter pylori (HpGnT) and β-1,4-galactosyltransferase of Neisseria meningitides (NmGalT), from Escherichia coli. We determined that use of magnesium (Mg2+) as a cofactor and at 55 oC, numerous sugar nucleotides were effectively synthesized in milligram-scale by RmlA in two hours, and these include uridine 5′-diphosphate galactose (UDP-Gal), uridine 5′-diphosphate N-acetylglucosamine (UDP-GlcNAc), uridine 5′-diphosphate glucose (UDP-Glc) and thymidine 5′-diphosphate glucose (TDP-glucose). Additionally, RmlA was site-specifically and covalently immobilized on an MNP using a combination of intein-mediated protein expression and NCL, and found that Rm1A-MNP retains almost 95% of its activity following ten consecutive enzyme assays. We also demonstrated synthesis of UDP-GlcNAc and UDP-Gal by using corresponding kinases from relatively cheap starting materials such as GlcNAc and Gal. All sugar nucleotides were purified by ion-exchange column for analytical purposes. The dissertation also demonstrates oligo-LacNAc synthesis in a cost-effective way. Normally, oligo-LacNAcs exist as an inseparable mixtures isomer in nature. By using our newly developed enzymatic system, defined lengths of oligo-LacNAcs were synthesized in a one-pot fashion by employing expressed NmGalT and HpGnT in the presence of UDP-Gal and UDP-GlcNAc. Also, we have demonstrated the versatility of the method by incorporating structurally more complex sialic acid residues with different linkages at the hitherto unknown internal Gal unit of oligo-LacNAc backbone in combination with α-2,3-sialyltransferase and α-2,6-sialyltransferase. Thus, we have achieved the synthesis of sialyl-oligo-LacNAcs; a hexa-saccharide with two repeating sialyl-LacNAc unit (4-26-2) and a nona-saccharide with three repeating sialyl-LacNAc unit (6-26-3), the attachment of which at the internal galactose unit was otherwise difficult by chemical means. With the enzymatic system, we can efficiently and quickly produce oligo-LacNAc derivatives. Finally to gain insights into the structure-activity studies, we have determined nine crystal structures of RmlA complexed with NDP-sugars, which we have synthesized enzymatically. Therefore, with the analysis of these structures, we can create amino acid mutation to improve the substrate tolerance and the catalytic efficiency of RmlA for accelerating progress in glycobiology.