CAD is a large, multifunctional protein, composed of three enzymatic domains: carbamoyl phosphate synthase II (CPS II), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO). The protein is involved in the three initial stages of de novo pyrimidine biosynthesis, required for the continuous production of pyrimidine nucleotides for living. In prokaryotes, the three enzymes are present in individual forms and acts independently. In eukaryotes, they are covalently linked together (~240 kDa) and form a homo-hexamer complex (~1.5 MDa) in the cellular environment. Up-regulation of CAD activity is often associated with the high demand of nucleotides during cell growth and proliferation; thus, the protein has been of an interest to many cancer biologists as a potential target for anti-tumour drugs.
CAD in yeast (Saccharomyces cerevisiae), alternatively called URA2, is a bi-functional protein that has CPS II and ATC activity only. DHO domain is substituted with non-functional pseudo-DHO (pDHO) instead, yet they are homologous to each other. We were interested to see whether the pDHO domain has an alternative role despite its inactivity. To do this, we expressed and purified yeast pDHO domain using bacterial system, and successfully crystallized and solved the structure at high resolution (1.2 Å). Despite many structural similarities with active DHO enzymes from other species, we were able to identify a number of differences that may account for the loss of enzymatic activity from the crystal structure. The biophysical properties were analysed via multi-angle light scattering (MALS), small angle x-ray scattering (SAXS), and negative-staining electron microscopy (EM). While majority successfully formed dimer, consistent with other active DHO enzymes, we observed the pDHO domain was also able to form hexamer in solution. This suggested that the inactive pDHO domain might play a critical structural role in full-length CAD forming homo-hexamer complex, facilitating efficient molecular tunnel mechanism in eukaryotic systems.