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 approximately 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 micromol/min/mg in the biosynthetic direction and 18.4 micromol/min/mg in the degradative direction, with K(m, carbamyl aspartate) = 323 microM and K(m, dihydroorotate) = 64.3 microM, 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.
Robles Lopez
Sonia M
SM
Department of Biochemistry, University of Puerto Rico School of Medicine, Medical Sciences Campus, San Juan, USA.
Hortua Triana
Miryam Andrea
MA
Zimmermann
Barbara H
BH
D000595:Amino Acid Sequence; D000818:Animals; D004080:Dihydroorotase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000493:pharmacokinetics; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015800:Protozoan Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism* / Q000493:pharmacokinetics; D016415:Sequence Alignment; D014122:Toxoplasma / Q000201:enzymology / Q000235:genetics*
D015800:Protozoan Proteins; D004080:Dihydroorotase