The effect of protein-tyrosine phosphorylation on an Escherichia coli K30 UDP-glucose dehydrogenase UgdK30
For many bacteria, capsular polysaccharides (CPS) are important virulence factors that allow the organism to withstand host defense mechanisms, such as phagocytosis and complement-mediated cell killing. CPS are polymers consisting of oligosaccharide repeat units, which are synthesized within the cytoplasm. Their assembly requires the concerted activity of several glycosyltransferases, as well as the enzymes necessary to produce the activated precursors specific to the repeat unit. For example, UDP-glucose dehydrogenase (Ugd) catalyses the conversion of UDP-glucose to UDP-glucuronic acid, and this serves as the precursor for uronic acids incorporated into the capsular repeat units of many groups of CPS in 'Escherichia coli'. Based on the evidence from a closely related homologue in an 'E. coli' K-12 strain (Ugd K-12), it was predicted that the Ugd from the group 1 capsule-producer, ' E. coli' K30 (UgdK30), might be phosphorylated by the tyrosine autokinase WZCK30. The activity of the kinase was already known to be required for CPS biosynthesis. This thesis describes an investigation of following hypothesis: A conserved regulatory mechanism involving tyrosine phosphorylation may exist involving UgdK30 and group 1 capsule production and export in an 'E. coli' K30 strain. Phosphorylation of UgdK30 by its cognate tyrosine kinase WZCK30 was confirmed 'in vitro' using purified proteins. Upon phosphorylation, UgdK30 exhibits a significant increase (3.2-fold) in catalytic activity. Further characterization of UgdK30 kinetic properties revealed that upon phosphorylation, UgdK30 exhibits a l.8-fold reduction in its K0.5 for UDP-glucose. Site-directed mutagenesis of several tyrosines in UgdK30 failed to identify a specific modified tyrosine involved in phosphorylation. The data suggest that the enzyme is phosphorylated at multiple residues. The 'in vivo' phosphorylation state of UgdK30 is still unclear. However, the data described in this thesis are consistent with the modification of Ugd K30 exerting a regulatory effect, potentially influencing CPSs biosynthesis.