Unraveling the physiological and metabolic impacts of a universal metabolite repair enzyme that removes a strong inhibitor of the TCA cycle

PROJECT SUMARY / ABSTRACT In general, enzymes are very precise at catalyzing a specific canonical reaction that fits within a particular metabolic network. Still, no enzyme is a perfect catalyst. The inherent flexibility of proteins makes it difficult for enzymes to distinguish their canonical substrate from structurally related compounds. Thus, many

enzymes act on unintended substrates (i.e., substrate promiscuity). Substrate promiscuities result in the formation of unintended or damaged metabolites (i.e., metabolite damage) that can be a useless drain on metabolism, and may be inhibitory and/or reactive, sometimes leading to toxicity. Accordingly, metabolite

damage repair enzymes exist for the specific purpose of counteracting metabolite damage, often by converting a damaged metabolite to a canonical one. The physiological importance of metabolite damage and its repair has been revealed over the past ~15-years as a handful of metabolic diseases in humans were

discovered to be caused by disruption of metabolite damage repair genes, many of which are highly conserved across the three domains of life. The proposed project will address metabolite damage repair associated to the TCA cycle – a universal core metabolic pathway that is involved in energy conversion and

is a source of chemical building blocks that supplies much of metabolism. The TCA cycle is a hotspot for metabolite damage due to high carbon flux through the pathway and chemical intermediates that are structurally similar organic acids, which can engage in promiscuous side reactions catalyzed by the abundant

cycle enzymes. I have identified and characterized several highly conserved metabolite damage control systems related to vitamin, cofactor, and amino acid metabolism, and my training has empowered me with a unique skillset and perspective that is allowing me to make similar discoveries related to the TCA cycle. One

enzyme that I have identified is particularly intriguing. A prevalent side-reaction of the TCA cycle enzyme succinate dehydrogenase oxidizes malate to enol-oxaloacetate (OAA), a metabolically inactive form of OAA that is a potent inhibitor of the TCA cycle. Our results provide strong evidence this side reaction is one of the

most prevalent promiscuous reactions in nature, and that enol-OAA is a potent inhibitor of the TCA cycle. We identified a universally conserved enzyme, OAT1, that removes the inhibitor, and show that bacterial cells lacking OAT1 have a severely attenuated TCA cycle. The proposed work will integrate biochemical,

genetic, and metabolomics/ fluxomics approaches to determine how OAT1 impacts the physiological and metabolic states of prokaryotic and single and multicellular eukaryotic model organisms. Completing this project will lead to the detailed characterization of a previously unrecognized but critical aspect of the TCA

cycle, ultimately redefining one of the most universal core metabolic pathways in biology. This work will also provide insights into mitochondrial metabolism and physiology that will impact human health and disease, and deliver a metabolite damage repair enzyme for use in optimizing synthetic biology platforms.