Directed evolution of the galactosyltransferase LgtC

Carbohydrates are known to play essential roles in various biological functions. The extremely diverse structures of oligosaccharides originate from the ability to combine different monosaccharides into glycans of varying length, sequence, and anomeric linkage. The classic chemical synthesis of complex oligosaccharides is a highly labor- intensive and time-consuming process, because of the wide range of regio- and stereo-chemical linkages, which require multiple protection and deprotection steps. Thus enzymatic synthesis represents an attractive alternative. Glycosyltransferases, the anabolic enzymes catalyzing glycan synthesis in nature, are ideal for oligosaccharide and glycopeptide synthesis due to their high regio- and stereospecific glycosidic linkage formation. Although many enzymes with different substrate specificities are known, the in vitro efficiency is often low and there are still many glycosidic linkages for which no biocatalyst is available so far. Enzyme engineering has an enormous potential for improving catalytic constants or altering substrate specificities and linkage formation for the synthesis of novel, non-natural and biologically relevant carbohydrate structures. This proposal is about the engineering of the lipopolysaccharyl-alpha-1,4-galactosyltransferase C (LgtC) from Neisseria meningitidis, which catalyzes the transfer of galactose from UDP-galactose to a terminal lactose. The product retains the configuration of the glycosidic bond of the donor sugar; the mechanism of these retaining glycosyltransferases is still discussed. A combination of rational enzyme engineering and directed evolution will be used to improve the catalytic properties of LgtC for the transfer of galactose to lactose and N-acetyllactosamine, respectively. First saturation mutagenesis will be used to alter amino acids in and around the active centre. As a second approach the gene is randomly mutagenized by error prone PCR. The mutated gene libraries will be expressed in E. coli and the transformants will be incubated with a fluorescence labelled acceptor sugar. Cells expressing LgtC with higher activity will show higher fluorescence and will be sorted by a fluorescence-activated cell sorter (FACS). Beneficial mutations will be identified by sequencing and the enzyme variants will be purified, characterized and additionally screened for acceptance of alternative donor sugars by thin layer chromatography. Another objective is the modification of the linkage formation of LgtC by screening the mutant libraries with labeled 4-fluorolactose as acceptor sugar. The blocked C-4 position allows no 1,4 transglycosylation activity, therefore only variants being able to form alternative linkages will result in higher fluorescence. The results of this study can provide an improved tool for oligosaccharide synthesis and additionally valuable information about the role of the investigated amino acid positions for the activity of this group of enzymes and therefore help to understand the until now unclear reaction mechanism as well as structure/function relations.