Directed evolution of a bacterial sialidase and characterization of mechanism based inactivation of glycosidases

Date created: 
2017-12-07
Identifier: 
etd10500
Keywords: 
Alpha glucosidase
Inhibition
Sialidase
Kinetics
Directed Evolution
Abstract: 

Sialic acids are often found at the terminal positions on the glycan chains that adorn all vertebrate cells and glycoproteins. This prominent position confers an essential role to sialic acid residues in biology, evolution and disease propagation. The most widespread sialic acid family members are N-acetylneuraminic acid, N-glycolylneuraminic acid and Kdn, which is an abbreviation for 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid. Enzymes that catalyze the removal of carbohydrate linkages from biological molecules are called glycoside hydrolases (GHs). These enzymes have been categorized into more than 130 different families. Glycoside hydrolase family 33 (GH33) contains exo-sialidases (E.C. 3.2.1.18, neuraminidases), from both eukaryotes and prokaryotes, which catalyze the hydrolysis of sialic acid from glycoconjugates. Interestingly, subtle differences exist in both the structure of the particular sialic acid and its position of attachment to glycoconjugate chains between humans and other mammals. These differences are indicators of the unique aspects of human evolution, and are relevant to understanding an array of human conditions. The present thesis reports on routes that we explored to further unravel the importance of sialic acids. We developed tools to probe for various sialic acid structures such as Kdn. To this end, we constructed a random mutant library of the neuraminidase from the soil bacterium Micromonospora viridifaciens (MvNA) and identified a number of recurring mutations in the sialidase gene which lead to a more efficient hydrolysis of synthetic natural substrate analogues such as 8FMU α-Kdn-(2→6)- β-D-Galp. We also using the available structure of wild type MvNA bound to the natural inhibitor, DANA, to identify amino acids potentially involved in recognition and binding to acetylated sialic acids and generated genetic libraries which we used along with positive and negative evolutionary screens to identify several clones capable of hydrolyzing Kdn glycosides more efficiently than Neu5Ac substrates. Kinetic studies on these clones allowed for determination of enzyme efficiencies and specificities. We also report our study of covalent inhibition of α-glucosidase from Saccharomyces cerevisiae (GH13). The measured pH-rate profiles for inhibition and reactivation as well as the corresponding catalytic and inhibitory proficiencies suggested that inhibition results from the formation of carbenium ions in the active site that are trapped rapidly by an enzymatic residue.

Document type: 
Thesis
Rights: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
File(s): 
Senior supervisor: 
Andrew Bennet
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) Ph.D.
Statistics: