Del Caño-Ochoa, FranciscoRamadane-Morchadi, LobnaEixerés, LluísMoreno-Morcillo, MaríaFernández-Leiro, RafaelRamón-Maiques, Santiago2024-12-182024-12-182024-12-01J Mol Biol . 2024 Dec 1;436(23):168832.https://hdl.handle.net/20.500.12105/25899The authors thank the clinicians, Drs. Paula Sanchez Pintos (Instituto de Investigacion Sanitaria Santiago de Compostela, La Coruna, Spain) and Joanna Kenny (Children's Health Ireland at Crumlin, Dublin, Ireland) for providing follow-up on the patients carrying the pathogenic variants reported in this study. This work was supported by grants PID2021-128468NB-I00 and PID2020-120258GB-I00 financed by MCIN/AEI/10.13039/501100011033 to SR-M and RF-L, respectively, and from Fundacion Ramon Areces Ciencias de la Vida (XX National Call) to SR-M, and Ramon y Cajal fellowship RYC-2017-23128 to RF-L. LRM was partially supported by the FIB-HCSC intramural young researcher mobility fellowship program 2023, FIB-HCSC, Madrid, Spain. X-ray diffraction experiments at synchrotrons were done through the participation of SR-M in the BAG proposals 2023077633 at ALBA, and MX-2452 at the European Synchrotron Radiation Facility (ESRF) with DOI10.15151/ESRF-ES-1117952942. The authors thank the ALBA and ESRF synchrotron staff for assistance during data collection.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.engVoRhttp://creativecommons.org/licenses/by-nc-nd/4.0/aspartate transcarbamoylasedihydroorotaseinborn errors of metabolismspathogenic variantpyrimidine nucleotide biosynthesisAttribution-NonCommercial-NoDerivatives 4.0 International3944767343623168832Journal of Molecular Biologyopen access