Bacterial peptidoglycan is a polymer made up of sugar chains, consisting of two alternating residues: N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Fragments of peptidoglycan containing chromophoric groups would be useful probes for studying glycosidases (such as lysozymes) that act on peptidoglycan. However, traditional synthesis of such fragments is difficult. Research has shown that mutant glycosidases can be designed that catalyze formation of glycosidic bonds when used in conjunction with appropriately activated donor sugars. We are making mutants of a glycosidase isolated from <i>Bacillus subtillus</i> (BSNagZ) to develop a glycosynthase capable of forming the NAG-NAM glycosidic bond. Mutagenesis of a plasmid containing the BSNagZ gene is being used to convert the nucleophilic catalytic residue Asp318 to Gly, Ala, and Ser. The resulting his-tagged proteins are expressed in <i>E. coli</i> and purified using a nickel-containing affinity column. The ability of these mutant enzymes to catalyze the coupling of a-1-fluoro-GlcNAc with 4-nitrophenyl N-acetyl-b-D-muramic acid will be examined.

Converting glycosidases to glycosynthases: Developing catalysts for synthesis of peptidoglycan fragments

2019 Spring National Meeting

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