Disruption of CAD Oligomerization by Pathogenic Variants

[Display omitted] •Pathogenic mutations disrupt CAD’s domain oligomerization and function.•Variant R1986Q inactivates CAD’s ATC domain and impairs trimerization.•Variant S1538L inactivates CAD’s DHO domain and impairs dimerization.•Assembly of CAD’s DHO dimers and ATC trimers are essential for in vivo function.•A model of CAD hexamer is generated integrating AlphaFold and crystal structures. CAD, the multi-enzymatic protein essential for initiating the de novo biosynthesis of pyrimidine nucleotides, forms large hexamers whose structure and function are not fully understood. Defects in CAD cause a severe neurometabolic disorder that is challenging to diagnose. We developed a cellular functional assay to identify defective CAD variants, and in this study, we characterized five pathogenic missense mutations in CAD’s dihydroorotase (DHO) and aspartate transcarbamoylase (ATC) domains. All mutations impaired enzymatic activities, with two notably disrupting the formation of DHO dimers and ATC trimers. Combining crystal structures and AlphaFold predictions, we modeled the hexameric CAD complex, highlighting the central role of the DHO and ATC domains in its assembly. Our findings provide insight into CAD’s stability, function, and organization, revealing that correct oligomerization of CAD into a supramolecular complex is required for its function in nucleotide synthesis and that mutations affecting this assembly are potentially pathogenic.