PROJECT SUMMARY/ABSTRACT Fatty acid β-oxidation (FAO) is a central metabolic pathway of great physiological relevance to human health and disease. FAO provides fuel for energy production, generates building blocks for the biosynthesis of many cellular molecules, detoxifies damaging lipids, and produces key signaling molecules. Disruption of this pathway contributes to fatty liver disease, hypoketotic hypoglycemia, obesity, insulin resistance, type 2 diabetes, and chronic kidney disease. Importantly, many fatty acids that ultimately are catabolized by FAO are not compatible with FAO until they have first been modified by other enzymes. These lipids include the 4-hydroxy fatty acids (4- HAs)—fatty acids that possess an oxidized carbon adjacent to the third, or β, position. Recent analyses empowered by advances in mass spectrometry have revealed a wide range of 4-HAs and 4-HA precursors in human plasma, which can originate from dietary intake, lipid peroxidation, and certain drugs of abuse. Prior analyses in liver revealed that 4-HAs are indeed processed by FAO once they are converted to compatible substrates. However, the enzymes responsible for this conversion are unknown, and the physiological relevance of these lipids is largely unexplored. Recently, we identified two atypical, highly uncharacterized FAO-related enzymes, acyl-CoA dehydrogenase 10 and 11 (ACAD10 and ACAD11), that appear capable of processing 4- HAs into FAO-friendly substrates via a novel mechanism. The long-term goals of this proposal are to define the roles of ACAD10 and ACAD11 in the catabolism of 4-HAs and to establish the metabolic ramifications that result from the disruption of these enzymes. We first aim to establish the specific biochemical reactions catalyzed by ACAD10 and ACAD11 in vitro through enzymology and structural biology approaches. We hypothesize that these ACADs employ their unique kinase domain, which is not found in other ACADs, to phosphorylate the 4- hydroxy position as part of their enzymatic mechanism. We then aim to establish the cellular locations and activities of each enzyme via microscopy and metabolite tracing studies. We hypothesize that ACAD10 is a mitochondrial enzyme responsible for the conversion of shorter-chain 4-HAs and that ACAD11 is a peroxisomal enzyme that converts longer-chain 4-HAs. Last, we aim to reveal the physiological ramifications of ACAD10 and ACAD11 disruptions in worms, mice, and humans. Notably, ACAD10 and ACAD11 have putative links to T2DM/insulin resistance, kidney disease, and coronary artery disease via GWAS studies, including in the Akimel O’odham (Pima Indian) tribe, and mouse models have linked ACAD10 and ACAD11 collectively to insulin resistance, weight gain, ectopic lipid deposition, and rhabdomyolysis; however, none of these have been studied directly. We hypothesize that the loss of these enzymes in mice, or the expression of specific ACAD10 variants in Pima Indians, will result in the accumulation of plasma 4-HA...