UBC Theses and Dissertations
Investigating the enzymatic mechanisms of the inverting and retaining glycosyltransferases by NMR spectroscopy Chan, Hau Wing
The overall goal of this thesis was to investigate the structures and enzymatic mechanisms of glycosyltransferases using NMR spectroscopy and enzyme kinetic measurements. The bifunctional sialyltransferase CstII from Campylobacter jejuni and the α-1,4-galactosyltransferase LgtC from Neisseria meningitidis were chosen to be the model inverting and retaining enzymes, respectively. By systematically introducing point mutations at the subunit interfaces of CstII, two active monomeric variants were obtained and characterized. In contrast to the wild-type tetramer, the monomeric CstII variants yielded good quality amide ¹H/¹⁵N-HSQC and methyl-TROSY NMR spectra. However, the absence of signals from approximately one half of the amides in the ¹H/¹⁵N-HSQC spectra of both monomeric forms suggests that the enzyme undergoes substantial conformational exchange on a msec-µsec time-scale. The histidine pKa values of CstII-F121D in its apo form were measured by monitoring the pH-dependent chemical shifts of biosynthetically incorporated [¹³Cε¹]-histidine. Consistent with its proposed catalytic role, the site-specific pKa value ~ 6.6 for His188 matches the apparent pKa value ~ 6.5 governing the pH-dependence of kcat/Km for CstII towards CMP-Neu5Ac. The enzymatic mechanism of the retaining glycosyltransferase LgtC appears to involve a “front-side attack” SNi or SNi-like mechanism with a short-lived oxocarbenium-phosphate ion pair intermediate. Furthermore, based upon X-ray crystallographic studies, two flexible loops were proposed to become ordered over the active site of LgtC upon sugar donor binding. Accordingly, NMR spectroscopy was used to investigate the dynamic properties of the enzyme with an emphasis on delineating the possible roles of these motions. The amide ¹H/¹⁵N-TROSY-HSQC and methyl-TROSY spectra of LgtC were partially assigned using a variety of NMR spectroscopic approaches, combined with mutagenesis of all the isoleucine residues. More than the expected number of methyl signals was observed, indicating that LgtC adopts multiple conformational states in equilibrium on a seconds time-scale, and that their relative populations change upon mutation and substrate binding. Furthermore, relaxation dispersion studies indicated substantial msec-µsec time-scale motions of methyl groups both within and distal to the active site in apo and substrate-bound forms of LgtC. Thus LgtC exhibits a range of dynamic behaviours potentially linked to its catalytic function. They were studies in this thesis using NMR spectroscopy and kinetic studies.
Item Citations and Data
Attribution-ShareAlike 3.0 Unported