Structure activity relationships of glycosyl transferases involved in protein O mannosylation in the actinobacteria

Actinobacteria have a protein O-glycosylation system that resembles eukaryotic protein Omannosylation. Both M. tuberculosis and S. coelicolor have growth retarded phenotypes when a membrane bound protein O-mannosyl transferase (Pmt), which transfers mannose from polyprenol phosphate mannose to a target protein, is absent. Moreover, S. coelicolor pmt – mutants are resistant to infection by ϕC31 phage and have increased susceptibility to vancomycin and several β-lactams. S. coelicolor strains that lack polyprenol phosphate mannose synthase (Ppm1), which transfers mannose from GDP-mannose to polyprenol phosphate, are even more susceptible to antibiotics and a ppm1 – mutant in M. tuberculosis is lethal. Pmt and Ppm1 are therefore possible new targets for the isolation of novel antimicrobials to be used against M. tuberculosis. The aim of this PhD thesis was to gain a deeper understanding of the structure and function of both S. coelicolor enzymes. For Ppm1, ten mutant alleles (the majority being individual alanine substitutions) were tested and eight were essential for S. coelicolor DT3017, a ppm1 – strain, complementation, with four of the corresponding residues positioned close to the predicted catalytic DXD motif. Attempts were made to purify and develop an enzyme assay for Ppm1 but we were overtaken by the activities from another laboratory (Manuela Tosin, Warwick) with whom we then collaborated. Enzyme activity data of detergent solubilised Ppm1 from the Tosin laboratory was in general agreement with my work on the in vivo phenotypes. Roles for these critical residues have consequently been proposed with a great degree of confidence, with the majority likely being catalytic and/or involved in substrate binding. Sequence alignments and structural bioinformatics were used with S. coelicolor Ppm1 and Pmt to identify targets for site-directed mutagenesis. Mutant alleles were introduced into ppm1 – (DT3017) or pmt – (DT2008/DT1025) S. coelicolor strains using conjugative integrative plasmids and scored for their ability to complement phage sensitivity and antibiotic hyper-susceptible phenotypes. Twenty-three highly conserved Pmt residues were each changed to alanine and six mutant alleles failed to complement the pmt- strains, indicating essentiality. Modelling the six corresponding residues indicated that five are positioned close to the predicted catalytic DE motif. Western blotting showed that none of the noncomplementing pmt – alleles expressed protein that could be localised to the membrane fractions and could not be detected in whole cell lysates. Conservative and semi-conservative substitutions were made to each of these six residues and with only a few exceptions, the alleles failed to complement DT1025 pmt –, indicating that strict conservation was necessary to preserve function. Moreover western blots continued to indicate that the non-complementing mutated Pmt proteins still failed to localise to the membrane. Evidently there is a relationship between the activity of Pmt and its localisation such that only functional Pmt could localise correctly to the membrane. Crucially, five of the six critical residues have not previously been proposed to have functional significance in the Pmt family and therefore could be important for the M. tuberculosis homologue. Whilst some of the mutations were predicted to impair catalytic activity, others may have affected localisation or substrate binding.