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Neisseria meningitidis lipopolysaccharide galactosyl transferase: mechanistic investigations and applications for oligosaccharide synthesis

University of British Columbia

The objective of this study was to characterise the activity of the recombinant Neisseria meningitidis UDP-galactose: 4-α-galactosyl transferase, lgtC-19, which has been expressed in Escherichia coli. Advances in cloning and expression techniques over the past decade have enabled access to a variety of glycosyl transferases not previously available in sufficient quantity for detailed exploration. This recent availability has led to a resurgence of interest in the behaviour of these enzymes and their potential applications in the enzymic synthesis of oligosaccharides. Although there has been a significant expansion in our understanding of these biological catalysts, much of this work has focused on the study of inverting enzymes and there is still relatively little information available regarding the kinetic and mechanistic behaviour of retaining nucleotide diphosphate hexosyl transferases. Additionally, there has not yet been reported a crystal structure for this class of enzyme, so that the structure of the active site has yet to be elucidated. LgtC-19 is a retaining α-galactosyl transferase, catalysing the transfer of galactose from UDP-galactose to lipopolysaccharide acceptors. In Nature, this enzyme recognises the reducing end of a lactose moiety as a suitable acceptor for its transfer reaction. The ability of this enzyme to recognise and accept a variety of alternative, synthetic glycosides as acceptor substrates as well as donor substrates has been investigated. These studies have shown lgtC- 19 to exhibit significant flexibility with regard to acceptor substrate structure. The fluorescent-labelled sugars FITC-lactose, FCHASE-lactose, and even FCHASE-galactose were capable of performing this function as were the simple disaccharide, lactose, and monosaccharide derivative, α-galactosyl fluoride. Although there have been other glycosyl transferases reported to utilise synthetic glycosides as acceptors, lgtC-19 appears to be the first such enzyme capable of using a simple glycoside as a donor substrate. The transferase activity of lgtC-19 was seen when α-galactosyl fluoride was provided as the glycosyl donor in the presence of catalytic amounts of UDP. This is the first report of any nucleotide diphosphate-dependent glycosyl transferase utilising a donor substrate which does not contain a nucleotide diphosphate functionality. With this flexibility, lgtC-19 shows excellent potential for use in the industrial-scale synthesis of oligosaccharides. The reaction conditions required by lgtC-19 were optimised by systematically altering the concentrations of substrates and co factors, investigating the effect of cofactors on enzyme activity, and varying the pH of enzyme storage solutions and of the assay itself. Once the conditions were optimised for maximum kinetic activity, the mechanism of enzyme action was investigated using Cleland's method. This analysis spanned a range of pH values and enabled the construction of pH curves for UDP-galactose, lactose, and α-galactosyl fluoride. From the information obtained through a variety of kinetic and mechanistic studies, the mechanism of galactosyl transfer employed by lgtC-19 was determined to be bi bi sequential.

Thesis/Dissertation

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2009-05-28

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http://hdl.handle.net/2429/8423

Chemistry

University of British Columbia

UBCV

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