title: Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis.
abstract: 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.
authors.affiliation: Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok, Thailand.
authors.forename: W
authors.initials: W
authors.lastname: Sirawaraporn
authors.forename: R
authors.initials: R
authors.lastname: Sirawaraporn
authors.forename: A
authors.initials: A
authors.lastname: Chanpongsri
authors.forename: W R
authors.initials: WR
authors.lastname: Jacobs
authors.forename: D V
authors.initials: DV
authors.lastname: Santi
mesh_terms: D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008969:Molecular Sequence Data; D009161:Mycobacterium / Q000187:drug effects / Q000201:enzymology*; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014296:Trimethoprim Resistance
chemical_list: D013762:Tetrahydrofolate Dehydrogenase