Year

2018

Degree Name

Doctor of Philosophy

Department

School of Chemistry

Abstract

Carbohydrate-active enzymes (CAZymes) are responsible for the synthesis, modification and degradation of glycosidic bonds in a large number of carbohydrate based structures. Among CAZymes, glycoside hydrolases (GH) and glycoside transferases (GT) catalyze the reversible breaking and formation of the glycosidic bonds, respectively. GHs have been shown to play vital roles in many biological processes. Consequently, they have drawn great attention as potential drug targets. This thesis will focus on a few critical issues in GHs. The Bacillus circulans xylanase (BcX) and Streptococcus pneumoniae (S. pneumoniae) sialidase are two of the important GH families, for which a great deal of information has been reported. At the same time there are critical mechanistic questions not well understood. BcX, a retaining GH11, contains a nucleophile Glu78 residue and a general acid/base Glu172 residue, which facilitates the glycosylation and deglycosylation step, respectively. A so-called “pKa cycling”, which is common in retaining GHs, was established for Glu172 of BcX. Various methods including quantum mechanics/molecular mechanics (QM/MM) free energy calculation, multiconformation continuum electrostatics (MCCE) and the PROPKA method were applied to predict the pKas or pKa shifts of Glu172 and other key residues, aiming to rationalise the effects of structural and electrostatic perturbations caused by mutations. S. pneumoniae sialidase, a GH33, is a common resident of the human nasopharynx that can cause a number of pathological conditions, including otitis media, meningitis and septicaemia. It encodes up to three distinct sialidases: NanA, a classical hydrolytic sialidase; NanB, an intramolecular tran-sialidase; and NanC, a sugar dehydratase. These three S. pneumoniae sialidases share a common first-step reaction pathway, while the second step shows an intriguing difference, producing Neu5Ac, 2,7-anhydro-Neu5Ac and Neu5Ac2en, respectively. The mechanisms, structural and energetic properties and the potential reaction pathways for NanA, NanB and NanC have been systematically studied using reaction barrier and energy calculations with combined QM/MM simulations. Complementary to previous studies, this thesis provided additional mechanistic insights into the determinants for their distinct reaction pathways.

FoR codes (2008)

0307 THEORETICAL AND COMPUTATIONAL CHEMISTRY, 0601 BIOCHEMISTRY AND CELL BIOLOGY

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.