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Investigations of micromonospora viridifaciens mutant sialidases in transglycosylations and novel enzymatic activity

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Thesis type
(Thesis) Ph.D.
Date created
Sialic acid is a monosaccharide with a nine-carbon atom backbone and there are more than fifty known derivatives of sialic acid depending on the substitution pattern, and these compounds make up the sialic acid family. The most widespread sialic acid family members are N-acetylneuraminic acid, N-glycolylneuraminic acid and Kdn. Sialic acid family members usually reside at the termini of sugar branch of glycoproteins and glycolipids in animals. As a result, the location of these sugars contributes to their roles in cellular and molecular recognition events. The present thesis focused on the biological mask function of sialic acid in the clearance and regulation of serum glycoconjugate lifetimes. Attaching sialic acid directly to terminal sugar residues can block the underlining galactose residues from recognition by the Ashwell receptors and tailor the glycoproteins clearance rate. Therefore, if sialic acid can be directly attached to recombinant glycoprotein drugs this will potentially decrease glycoprotein clearance rates; thereby allowing the glycoprotein drug’s dosage to be reduced. The present thesis focused on studying the ability of mutant Micromonospora viridifaciens sialidases to transfer sialic acid onto other sugar acceptors. The wild-type sialidase favors hydrolysis rather than transfer of sialic acid. We show that the Y370G mutant sialidase is flexible in transferring sialic acid onto several different sugar acceptors. To try and improve the yield for the transglycosylation reaction of the Y370G mutant sialidase, hydrophobic loops, which were incorporated on the enzyme to try and increase the local concentration of acceptor. The resultant loop mutants abolished the enzyme’s activity. Therefore, we targeted mutagenesis of the loop region to create a library of Y370G-loop mutants. Unfortunately, screening of this mutant library did not yield any promising next generation Y370G mutants. We also screened another mutant library based on two tyr370 nucleophile mutants, histidine (Y370H) and methionine (Y370M), with the goal of finding novel mechanisms of action. However, screening of this library produced several revertant mutants, but no promising candidates with either histidine or methionine as the enzymatic nucleophile.
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Scholarly level
Supervisor or Senior Supervisor
Thesis advisor: Bennet, Andrew
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