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 isolated and expressed in Escherichia coli, and the enzyme has been purified and characterized. The coding sequence of the DHFR-TS is 1581 nt, encoding a 527-amino-acid protein of 58,505 Da. The gene was expressed under control of the trc promoter in pKK233-2. The resulting expression plasmid conferred trimethoprim resistance to E. coli DH5 alpha and complemented the TS deficiency in chi 2913recA cells indicating the presence of active DHFR and TS. DHFR-TS was purified by methotrexate-Sepharose chromatography. In addition to the full-length enzyme, the purified enzyme contained 31 and 31.5-kDa forms of the enzyme that cross-reacted 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 presence of sufficient TS for complementation, TS activity was not detectable in the crude extract or in the final purified enzyme preparation. Although the majority of the enzyme appears to be full length, it is possible 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 kcat and Km values similar to those of related enzymes. The T. brucei DHFR has Ki values for antimicrobial antifolates pyrimethamine and trimethoprim which are significantly lower than the closely related T. cruzi or L. major DHFRs or than human DHFR.
Gamarro
F
F
Department of Biochemistry and Biophysics, University of California, San Francisco 94143, USA.
Yu
P L
PL
Zhao
J
J
Edman
U
U
Greene
P J
PJ
Santi
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D004790:Enzyme Induction; D005493:Folic Acid Antagonists / Q000494:pharmacology; D017125:Genes, Protozoan; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D011739:Pyrimethamine / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000378:metabolism; D013940:Thymidylate Synthase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D014295:Trimethoprim / Q000494:pharmacology; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics*; D014349:Trypanosoma cruzi / Q000235:genetics
D005493:Folic Acid Antagonists; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D011739:Pyrimethamine