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Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis. 1991

W Sirawaraporn, and R Sirawaraporn, and A Chanpongsri, and W R Jacobs, and D V Santi
Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc2(6) and trimethoprim-resistant mutant mc2(26) was purified to homogeneity. In crude extracts, the specific activity of the enzyme from the trimethoprim resistant strain was comparable to that from the sensitive strain. The DHFR from both sources was purified using affinity chromatography on MTX-Sepharose followed by Mono Q FPLC. The enzyme has an apparent molecular mass of 23 kDa from gel filtration on Sephadex G-100 and from SDS-PAGE. Amino terminal sequence analysis showed homology with DHFRs from a subset of other gram-positive organisms. The purified enzyme from the trimethoprim-sensitive organism exhibited Km values for H2folate and NADPH of 0.68 +/- 0.2 microM and 21 +/- 4 microM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 +/- 0.4 microM and 5.3 +/- 1.5 microM, respectively. A kcat of 4.5 sec-1 was determined for the DHFR from both sources. The enzyme from both sources was competitively inhibited by pyrimethamine and trimethoprim. The Ki value of trimethoprim, for the enzyme from the drug-resistant organism was about six-fold higher than for the enzyme from drug-sensitive strain. Our data suggest that mutation of DHFR contributes to trimethoprim resistance in the mc2(26) strain of M. smegmatis.

UI MeSH Term Description Entries
D007700 Kinetics The rate dynamics in chemical or physical systems.
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009161 Mycobacterium A genus of gram-positive, aerobic bacteria. Most species are free-living in soil and water, but the major habitat for some is the diseased tissue of warm-blooded hosts. Mycobacteria
D002846 Chromatography, Affinity A chromatographic technique that utilizes the ability of biological molecules, often ANTIBODIES, to bind to certain ligands specifically and reversibly. It is used in protein biochemistry. (McGraw-Hill Dictionary of Scientific and Technical Terms, 4th ed) Chromatography, Bioaffinity,Immunochromatography,Affinity Chromatography,Bioaffinity Chromatography
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D000595 Amino Acid Sequence The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION. Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D013762 Tetrahydrofolate Dehydrogenase An enzyme of the oxidoreductase class that catalyzes the reaction 7,8-dihyrofolate and NADPH to yield 5,6,7,8-tetrahydrofolate and NADPH+, producing reduced folate for amino acid metabolism, purine ring synthesis, and the formation of deoxythymidine monophosphate. Methotrexate and other folic acid antagonists used as chemotherapeutic drugs act by inhibiting this enzyme. (Dorland, 27th ed) EC 1.5.1.3. Dihydrofolate Dehydrogenase,Dihydrofolate Reductase,Folic Acid Reductase,Acid Reductase, Folic,Dehydrogenase, Dihydrofolate,Dehydrogenase, Tetrahydrofolate,Reductase, Dihydrofolate,Reductase, Folic Acid
D014296 Trimethoprim Resistance Nonsusceptibility of bacteria to the action of TRIMETHOPRIM.

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