Cloning and preliminary characterization of the dihydroorotase from Toxoplasma gondii

A full-length dihydroorotase (DHOase) sequence was cloned from a Toxoplasma gondii tachyzoite cDNA library. The sequence had a calculated molecular mass of 44.2 kDa and a pI of 5.72, and was most similar to type IIa DHOases. A recombinant protein was expressed and purified with a yield of similar to 20 mg L-1 of cell culture. Polyclonal antibodies raised against purified recombinant protein reacted with a band of the expected molecular mass in tachyzoite extracts. Specific activities of 18.3 mu mol/min/mg in the biosynthetic direction and 18.4 mu mol/min/mg in the degradative direction, with K-m,K- carbamyl aspartate = 323 mu M and K-m,K- dihydroorotate = 64.3 mu M, were measured for purified recombinant protein. Size exclusion chromatography/laser light scattering showed a single, monodisperse peak with a molecular mass of 45.6 kDa, suggesting that the native protein is a monomer. (c) 2006 Elsevier B.V. All rights reserved.