HETEROLOGOUS EXPRESSION AND CHARACTERIZATION OF THE BIFUNCTIONAL DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE ENZYME OF TOXOPLASMA-GONDII

We have expressed catalytically active Toxoplasma gondii dihydrofolate -thymidylate synthase (DHFR-TS) and the individual TS and DHFR domains in Escherichia coil using the T7 promoter of pET-15b. DHFR-TS constit uted approximately 10% of the total soluble cell protein and was purif ied using methotrexate-Sepharose chromatography to yield 10 mg of homo geneous DHFR-TS per liter of culture. The DHFR domain was recovered as insoluble inclusion bodies which could be unfolded and refolded to re cover soluble, active enzyme, The TS domain was overexpressed as a sol uble protein by growing the cells at 24 degrees C; this is the first r eport of the expression of an active TS domain from a bifunctional enz yme. The k(cat) and K-m values for DHFR-TS are similar to those of oth er previously characterized protozoan DHFRs and TSs, The antimicrobial antifolates, TMP and Pyr, inhibit DHFR activity of the bifunctional p rotein in accord with their effects in crude enzyme preparations and i n vivo systems, Kinetic parameters and Ki values for TMP and Pyr with the isolated DHFR domain were identical to the values for DHFR in the bifunctional enzyme. Evidence of kinetic channeling of the dihydrofola te product of TS to the DHFR domain in the bifunctional enzyme was obt ained by kinetic and inhibition studies, Properties such as yield, sta bility, and activities of the recombinant T. gondii DHFR-TS provide cl ear advantages over other bifunctional DHFR-TSs as a model for future studies.