Abstract

Short-chain acyl-CoA dehydrogenase (SCAD) is a critical enzyme in mitochondrial fatty acid β-oxidation, catalyzing the initial dehydrogenation of short-chain acyl-CoAs. Mutations in theACADSgene cause SCAD deficiency (SCADD), a disorder with remarkably heterogeneous clinical presentation. However, the molecular mechanisms underlying substrate specificity and the pathogenicity of mostACADSvariants remain poorly understood. Here, we present high-resolution cryo-EM structures of human SCAD in complex with its physiological substrate butyryl-CoA (C4) and the longer substrate hexanoyl-CoA (C6). The butyryl-CoA-bound structure at 2.1 Å resolution details a pre-catalytic geometry ideal for hydride transfer, with Glu392 positioned as the catalytic base. We systematically characterized nineteen disease-associated mutations, which we classify into three functional categories: those disrupting FAD binding, those impairing substrate binding, and those compromising protein folding and stability. In addition, using the W177R mutant as a representative model, we demonstrate that folding-defective mutations provoke protein aggregation, leading to proteotoxicity, oxidative stress, and apoptosis, revealing a pathogenic mechanism beyond mere catalytic loss. In brief, our integrated findings elucidate the structural determinants of substrate specificity and catalytic mechanism in SCAD, and provide mechanistic insights into the functional impairments caused by mutations linked to SCADD.