Characterization of enzymes that modify or degrade the Pseudomonas virulence factor, alginate

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Montana State University - Bozeman, College of Agriculture


Biosynthesis of the polysaccharide alginate is important for Pseudomonas aeruginosa to establish chronic pulmonary infections in Cystic Fibrosis patients. Alginate is a linear polymer of β 1-4 linked D-mannuronate (M) interspersed with its C-5 epimer, L-guluronate (G). Initially D-mannuronate residues are polymerized into the periplasm as polymannuronic acid. In the periplasm, some polymannuronate residues are converted to L-guluronate residues by the C-5 epimerase, AlgG. Alginate is further modified by the addition of O-acetyl groups to the D-mannuronate residues Algl, AlgJ, and AlgF. The focus of this research was to further characterize the alginate modifying enzymes, AlgG and AlgJ. We found that AlgG contains a repeating sequence that is characterized as a CArbohydrate-binding and Sugar Hydrolases (CASH) domain. Proteins containing this domain fold as right-handed β-helices (RHβH) and bind to long chain linear polysaccharides. AlgG was predicted to fold as a RHβH by the 3D-PSSM structural prediction program. RHβH models of AlgG predict that the identified 324-DPHD-327 motif lies in the long shallow groove that may accommodate alginate. Site-directed mutations of this motif disrupt enzymatic activity, but not structural integrity, suggesting that these mutations lie in the epimerase catalytic domain. Asparagines 362 and 367 are predicted to stack with other asparagine residues along the β-helix. Results obtained from site directed mutants of N362 or N367 suggest that these mutations disrupt asparagine stacking and protein stability. Original attempts to identify alginate binding motifs were made using phage display peptide libraries. This technique proved unsuccessful in identifying binding motifs in AlgG or AlgJ, as discussed in Chapter 3. However, we were able to characterize AlgG with structural modeling, and identified two potentially important motifs in AlgJ. Two putative guluronate specific lyases were also identified in P. aeruginosa, PA1167 and PA1784. Overexpression of PA1167 in mucoid strains FRD1 and FRD1153 results in a non-mucoid phenotype, suggesting this acts as an alginate lyase. The experiments also show the P. aeruginosa cannot use alginate as a carbon source. This research provides a greater understanding of carbohydrate/protein interactions between alginate modifying enzymes and alginate.




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