Carbamoyl phosphate synthetase II (CPS II), aspartate transcarbamoylase (ATC), and dihydroorotase (DHO) are three enzymes involved in the first three steps of de novo pyrimidine biosynthesis. In bacteria, these proteins are present in individual enzymes and function independently; however, in eukaryotes they are often found in a covalently linked form. In mammals, the enzymes are present as a large, multienzyme complex called CAD (CPS II-ATC-DHO, 243 kDa) and this complex has been proposed to form a homo-hexamer of ~1.5 MDa in the cellular environment. It is known that over-expression of CAD is required for proliferation and is associated with tumour cell development; thus, understanding the structural basis of the complex will be useful in cancer biology.

Although crystal structures are available for the individual enzymes in prokaryotes, little is known about the assembly and regulation of the eukaryotic CAD complex. Thus, this project aims to study the structures of:

1) Native form of eukaryotic CAD complexes in assembled and functional state, and

2) Each domain present in eukaryotic CAD in recombinant form

The structural analysis of the complex will be performed using both electron microscopy (EM) and crystallography. The ultimate aim is to generate a pseudo-high resolution model from a combination of these two approaches.