# Structure and Substrate Specificity of Human Short-Chain Acyl-CoA Dehydrogenase and Insights into Pathogenicity of Disease-Associated Mutations

## TL;DR

This study reveals the structure of SCAD enzyme and explains how mutations cause disease by affecting its function and stability.

## Contribution

High-resolution SCAD structures and classification of disease mutations into functional categories with mechanistic insights.

## Key findings

- Cryo-EM structures show SCAD's pre-catalytic geometry and role of Glu392 in hydride transfer.
- Nineteen disease mutations are grouped into FAD binding, substrate binding, and folding/stability defects.
- Folding-defective mutations like W177R cause aggregation, proteotoxicity, oxidative stress, and apoptosis.

## 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 the ACADS gene cause SCAD deficiency (SCADD), a disorder with remarkably heterogeneous clinical presentation. However, the molecular mechanisms underlying substrate specificity and the pathogenicity of most ACADS variants 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.