TRYPANOSOMA-BRUCEI DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE - GENE ISOLATION AND EXPRESSION AND CHARACTERIZATION OF THE ENZYME

The gene encoding the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) of Trypanosoma brucei brucei has been isolat ed and expressed in Escherichia coli, and the enzyme has been purified and characterized. The coding sequence of the DHFR-TS is 1581 nt, enc oding a 527-amino-acid protein of 58505 Da. The gene was expressed und er control of the trc promoter in pKK233-2. The resulting expression p lasmid conferred trimethoprim resistance to E. coli DH5 alpha and comp lemented the TS deficiency in (chi)2913recA cells indicating the prese nce of active DHFR and TS. DHFR-TS was purified by methotrexate-Sephar ose chromatography. In addition to the full-length enzyme, the purifie d enzyme contained 31 and 31.5-kDa forms of the enzyme that cross-reac ted with anti-L. major DHFR-TS antibodies; one was truncated at the N- and C termini, and the other at only the C terminus. Despite the pres ence of sufficient TS for complementation, TS activity was not detecta ble in the crude extract or in the final purified enzyme preparation. Although the majority of the enzyme appears to be full length, it is p ossible that the TS domain has been degraded by one of more residues, which would inactivate the ability to synthesize thymidylate. Kinetic analysis of DHFR yielded k(cat) and K-m values similar to those of rel ated enzymes. The T. brucei DHFR has K-i values for antimicrobial anti folates pyrimethamine and trimethoprim which are significantly lower t han the closely related T. cruzi or L. major DHFRs or than human DHFR.