Molecular cloning, recombinant expression and partial characterization of the aspartate transcarbamoylase from Toxoplasma gondii

A cDNA coding for a monofunctional aspartate transcarbamoylase (ATCase) was isolated from a Toxoplasma gondii tachyzoite cDNA library using a complementation method. The calculated molecular mass of the deduced amino acid sequence was 46.8 kDa, with a predicted pI of 7.1. Size exclusion chromatography/laser-light scattering showed a single, monodisperse peak with molecular mass of 144 kDa. Amino acid sequence alignments revealed that active site residues of the Escherichia coli ATCase catalytic chain were conserved in the T. gondii sequence, and the latter shared 26-33% overall sequence identity with other ATCases. A recombinant enzyme was overexpressed in E. coli, and was purified with a yield of similar to0.8 mg l(-1) culture. The temperature dependence of the recombinant enzyme was similar to that of native ATCase in T. gondii extracts. The K-m's for aspartate and carbamoyl phosphate were 7.82 mM, and 67.6 muM, respectively. The V-max was 23 900 mumol h(-1) mg(-1). Pyrimidine nucleotides had no significant effect on the enzyme's activity. N-phosphonoacetyl-L-aspartate (PALA) inhibited the enzyme with K-i = 0.38 muM. The T. gondii ATCases contained two additional sequences of similar to24 residues each, which are not found in other ATCases. One of these sequences was susceptible to proteolysis by elastase. (C) 2002 Elsevier Science B.V. All rights reserved.