Functional expression of the dihydrofolate reductase domain of Leishmania major dihydrofolate reductase-thymidylate synthase bifunctional protein.
The dihydrofolate reductase (DHFR) domain of the bifunctional dihydrofolate reductase-thymidylate synthase from Leishmania major has been subcloned and expressed as a soluble protein in Escherichia coli strain PA414 harboring plasmid pLMDHFR. Homogeneous L. major DHFR was obtained by chromatography on methotrexate-Sepharose followed by DE52. The purified enzyme migrated as a single 25-kDa protein on SDS-PAGE. The native molecular weight was determined to be 26 kDa, indicating that the isolated domain is a monomer. N-terminal sequence analysis revealed that serine, the second amino acid in the coding sequence, was the N-terminal amino acid of the protein. The enzyme showed a pH optimum similar to that of the bifunctional protein. For purified DHFR, the Km values were <1.0 microM for H2folate and <1.0 microM for NADPH. The kcat of the most active DHFR preparation was 5 s-1. The Km and kcat values were similar to those of the bifunctional enzyme.
Yu
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PL
Department of Biochemistry and Biophysics and Pharmaceutical Chemistry, University of California, San Francisco, 94143-0446, USA.
Zhao
J
J
Yu
M
M
Reid
R
R
Santi
D V
DV
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D017124:Conserved Sequence / Q000235:genetics; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D007700:Kinetics; D018320:Leishmania major / Q000201:enzymology*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013940:Thymidylate Synthase / Q000378:metabolism*
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase