Probing conformational states of glutaryl-CoA dehydrogenase by fragment screening

Glutaric acidemia type 1 is an inherited metabolic disorder which can cause macrocephaly, muscular rigidity, spastic paralysis and other progressive movement disorders in humans. The defects in glutaryl‐CoA dehydrogenase (GCDH) associated with this disease are thought to increase holoenzyme instability and reduce cofactor binding. Here, the first structural analysis of a GCDH enzyme in the absence of the cofactor flavin adenine dinucleotide (FAD) is reported. The apo structure of GCDH from Burkholderia pseudomallei reveals a loss of secondary structure and increased disorder in the FAD‐binding pocket relative to the ternary complex of the highly homologous human GCDH. After conducting a fragment‐based screen, four small molecules were identified which bind to GCDH from B. pseudomallei. Complex structures were determined for these fragments, which cause backbone and side‐chain perturbations to key active‐site residues. Structural insights from this investigation highlight differences from apo GCDH and the utility of small‐molecular fragments as chemical probes for capturing alternative conformational states of preformed protein crystals.