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 coli using the T7 promoter of pET-15b. DHFR-TS constituted approximately 10% of the total soluble cell protein and was purified using methotrexate-Sepharose chromatography to yield 10 mg of homogeneous DHFR-TS per liter of culture. The DHFR domain was recovered as insoluble inclusion bodies which could be unfolded and refolded to recover soluble, active enzyme. The TS domain was overexpressed as a soluble protein by growing the cells at 24 degrees C; this is the first report of the expression of an active TS domain from a bifunctional enzyme. The kcat and K(m) values for DHFR-TS are similar to those of other previously characterized protozoan DHFRs and TSs. The antimicrobial antifolates, TMP and Pyr, inhibit DHFR activity of the bifunctional protein in accord with their effects in crude enzyme preparations and in 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 dihydrofolate product of TS to the DHFR domain in the bifunctional enzyme was obtained by kinetic and inhibition studies. Properties such as yield, stability, and activities of the recombinant T. gondii DHFR-TS provide clear advantages over other bifunctional DHFR-TSs as a model for future studies.
Trujillo
M
M
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Donald
R G
RG
Roos
D S
DS
Greene
P J
PJ
Santi
D V
DV
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015870:Gene Expression; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011739:Pyrimethamine / Q000494:pharmacology; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014122:Toxoplasma / Q000201:enzymology* / Q000235:genetics; D014295:Trimethoprim / Q000494:pharmacology
D017931:DNA Primers; D016054:DNA, Protozoan; D005493:Folic Acid Antagonists; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; C056930:thymidylate synthase-dihydrofolate reductase; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; D011739:Pyrimethamine