The proposed research program is a detailed investigation into the enzymology of alginate biosynthesis in the pathogenic bacterium Pseudomonas aeruginosa. P. aeruginosa infections are common and present significant health hazards to humans. Complications arising from colonization of lung tissues by P. aeruginosa are the leading cause of morbidity and mortality in cystic fibrosis patients. Alginate is a linear polysaccharide composed of mannuronate and guluronate residues, and is secreted by the bacteria to form an extracellular capsule, which contributes to their ability to effectively colonize lung tissue, resist antibiotic therapies and evade the host's immune system response. A potential strategy to combat P. aeruginosa infections is to develop agents which inhibit alginate biosynthesis and thereby render the bacteria susceptible to conventional antibiotics. Reports in the literature suggest that this strategy has merit, but to date, there are no effective specific inhibitors or inactivators of P. aeruginosa alginate biosynthetic enzymes. In order to approach the inhibition of alginate biosynthesis in a rational way, a deeper understanding of the functional properties and catalytic mechanisms of the constituent enzymes of the pathway is required. The research program described in the proposal focuses on C5 mannuronan epimerase and the enzymes which catalyze the first four steps of the alginate biosynthetic pathway. GDP-mannose dehydrogenase catalyzes the committed step in alginate biosynthesis, a mechanistically interesting four-electron oxidation, and will receive particularly close scrutiny. Detailed kinetic studies using transient kinetic approaches and kinetic isotope effect measurements will be performed in order to determine the energetics of the reactions; potential inhibitors and inactivators which have been designed based on hypotheses about the enzyme's chemical mechanisms will be characterized. The structure of phosphomannomutase, which catalyzes the second step in the pathway, will be determined by X-ray crystallography; GDP-mannose dehydrogenase has also been crystallized, and the determination of its structure will be pursued.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physical Biochemistry Study Section (PB)
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Marino, Pamela
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University of Missouri-Columbia
Schools of Medicine
United States
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Fast, Walter; Tipton, Peter A (2012) The enzymes of bacterial census and censorship. Trends Biochem Sci 37:7-14
Raychaudhuri, Aniruddha; Tullock, Amanda; Tipton, Peter A (2008) Reactivity and reaction order in acylhomoserine lactone formation by Pseudomonas aeruginosa RhlI. Biochemistry 47:2893-8
Jerga, Agoston; Raychaudhuri, Aniruddha; Tipton, Peter A (2006) Pseudomonas aeruginosa C5-mannuronan epimerase: steady-state kinetics and characterization of the product. Biochemistry 45:552-60
Jerga, Agoston; Stanley, Matthew D; Tipton, Peter A (2006) Chemical mechanism and specificity of the C5-mannuronan epimerase reaction. Biochemistry 45:9138-44
Regni, Catherine; Schramm, Andrew M; Beamer, Lesa J (2006) The reaction of phosphohexomutase from Pseudomonas aeruginosa: structural insights into a simple processive enzyme. J Biol Chem 281:15564-71
Naught, Laura E; Tipton, Peter A (2005) Formation and reorientation of glucose 1,6-bisphosphate in the PMM/PGM reaction: transient-state kinetic studies. Biochemistry 44:6831-6
Kimmel, Jennifer L; Tipton, Peter A (2005) Inactivation of GDP-mannose dehydrogenase from Pseudomonas aeruginosa by penicillic acid identifies a critical active site loop. Arch Biochem Biophys 441:132-40
Raychaudhuri, Aniruddha; Jerga, Agoston; Tipton, Peter A (2005) Chemical mechanism and substrate specificity of RhlI, an acylhomoserine lactone synthase from Pseudomonas aeruginosa. Biochemistry 44:2974-81
Liu, Hao-Yang; Wang, Ziaohui; Regni, Catherine et al. (2004) Detailed kinetic studies of an aggregating inhibitor; inhibition of phosphomannomutase/phosphoglucomutase by disperse blue 56. Biochemistry 43:8662-9
Raychaudhuri, Aniruddha; Tipton, Peter A (2004) Protocol for amplification of GC-rich sequences from Pseudomonas aeruginosa. Biotechniques 37:752, 754, 756

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