Toxoplasma gondii: Molecular cloning and characterization of a nitric oxide synthase-like protein

Toxoplasma gondii has a nitrite production and a putative nitric oxide synthase (NOS) motif genomic sequence. In order to demonstrate that this sequence is functional and could be involved in the metabolism of l-arginine derivatives, we constructed a baculovirus carrying the previously identified Toxoplasma NOS-like DNA sequence. The recombinant protein was expressed into insect Sf9 cells and his activity was tested in serial microplate colorimetric assays. The protein produced 21 nmol/min/ml nitrites per microgram of protein and followed Michaelis–Menten kinetics, with a Km for l-arginine of 2.3 mM. Furthermore, the optimal pH, temperature and incubation time for the recombinant Toxoplasma NOS-like protein were established. Toxoplasma NOS runs as a band of 11.6 kDa on tricine–sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Our results indicate that the recombinant protein derived from the putative genomic sequence, at the chromosome 1b of T. gondii, is able to produce nitrites from l-arginine as substrate.