Bacteria use numerous small molecules for cellular signaling. These primary and secondary messengers act within the cell to relay signal transduction and regulate distinct pathways. Among these the diatomic gas molecule nitric oxide (NO) and the nucleotide cyclic-di-GMP play central role in virulence, quorum sensing, and biofilm formation. In this dissertation, we have focused on H-NOX, a heme-nitric oxide/oxygen binding protein in the biofilm-dwelling bacterium Shewanella woodyi (Sw), which mediates NO-induced biofilm dispersal by modulating the activity of a dual-functioning diguanylate cyclase/phosphodiesterase enzyme that we have named HaCE, (H-NOX-associated cyclic-di-GMP enzyme). These enzymes tightly regulate the intracellular spatio-temporal concentrations of cyclic-di-GMP which is synthesized from 2 molecules of GTP by enzymes called Diguanylate Cyclases (DGC), and gets hydrolyzed to pGpG by enzymes called Phosphodiesterases (PDE), in turn controlling biofilm formation. Thus, H-NOX/HaCE represents a potential drug target for regulating biofilm formation. This is the first biophysical and structural study of an NO-bound SwH-NOX/SwHaCE complex. We have shown that SwH-NOX/SwHaCE associate in a α 2β2 (heterotetramer) stoichiometry. The SwH-NOX surface residues critical for binding to SwHaCE have been identified using NMR studies. Fluorescent quenching binding studies, co-immunoprecipitation and enzyme assays confirm this protein-protein interface and its importance for H-NOX/HaCE function.
Also described is the role of charged residues that are required for substrate binding and divalent metal ion coordination in the hydrolysis of cyclic-di-GMP by PDE domains found in bi-functional enzymes with an N-terminal DGC domain. Crystal structures of active PDE enzymes indicate a TIM-barrel type of fold. The catalytic pocket, situated towards the C-terminus, contains an unstructured loop, called "loop 6", which is conserved in this family of enzymes. The role of these conserved residues has been elucidated using mutational studies combined with biophysical studies and enzymatic analysis to show how structure affects enzyme function.
|Advisor:||Boon, Elizabeth M.|
|Commitee:||Rizzo, Robert C., Thanassi, David G., Tonge, Peter J.|
|School:||State University of New York at Stony Brook|
|School Location:||United States -- New York|
|Source:||DAI-B 75/04(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Organic chemistry|
|Keywords:||Cyclic-di-GMP, Nitric oxide, Shewanella woodyi|
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