Sirtuins and Metabolic Pathway Integration

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DESCRIPTION (provided by applicant): The long term goal of this work is to understand the contributions of the sirtuin-dependent protein acetylation/deacetylation system (SDPADS) to prokarybtic cell physiology, in particular to metabolic pathway integration. In the initial funding period of GM62203 we reconstituted the entire propionate catabolism in vitro using purified components. We also determined that propionyl-CoA, an intermediate in propionate catabolism, is the metabolite that integrates the catabolic pathways for propionate and 1,2-propanediol, and established that 2-methylcitrate is a potent inhibitor of cell growth. We also uncovered an exciting link between acetate and propionate degradation and the protein deacetylases known as sirtuins. Sirtuins are conserved in all forms of life from prokaryotes to humans, and play a key role in cell aging and age-related diseases making the research proposed here relevant to human health. Sirtuins play a role in eukaryotic gene expression, a role yet to be established in prokaryotes. We also identified the protein acetyltransferase enzyme which together with sirtuin, comprise the SDPADS. Salmonella enterica is an excellent system for studying the integration of energy generation and carbon utilization pathways in prokaryotes by the SDPADS. The identification of a link between metabolism and sirtuin function raised many exciting questions about the role of the SDPADS in cell physiology. However, before addressing broad questions we need to understand how the system works mechanistically. Here we propose to begin characterizing the SDPADS of S. enterica. We seek answers to basic questions about the SDPADS, such as: What are the protein substrates of the SDPADS? How is SDPADS function regulated by the cell? How do the acetylase or deacetylase enzymes interact with their protein substrates? What is the fate of the metabolite product of the deacetylase reaction? Comprehensive genetic, molecular biological, biochemical and structural approaches will be taken to answer these fundamental questions of prokaryotic cell physiology.