Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase.
The gene for dihydrofolate reductase of Mycobacterium tuberculosis was amplified by polymerase chain reaction (PCR) from M. tuberculosis H37Rv strain genomic DNA. The protein was expressed in inclusion bodies in high yield in Escherichia coli under the control of the T7 promoter. Active enzyme was obtained by refolding from guanidine HCl and after a single chromatography step the sample was > 99% homogeneous with a specific activity of approximately 15.5 micromol min(-1) mg(-1). Mass spectrometry analysis confirmed the expected mass of 17.6 kDa. Gel filtration of the enzyme indicated that it was a monomer. Steady-state kinetic parameters were determined and the effect of pH and KCl on the enzyme examined. Methotrexate and trimethoprim inhibited the enzyme.
White
E Lucile
EL
Drug Discovery Division, Southern Research Institute, 2000 Ninth Avenue South, Birmingham, AL 35205, USA. white@sri.org
Ross
Larry J
LJ
Cunningham
Amanda
A
Escuyer
Vincent
V
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics*; D011189:Potassium Chloride; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D011189:Potassium Chloride; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate