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

Worachart Sirawaraporn, Rachada Sirawaraporn, Atid Chanpongsri, William R. Jacobs, Daniel V. Santi

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

Dihydrofolate reductase (DHFR) from extracts of Mycobacterium smegmatis strain mc26 and trimethoprim-resistant mutant mc226 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 μM and 21 ± 4 μM, respectively. The Km values for H2folate and NADPH for the enzyme from the drug-resistant organism were 1.8 ± 0.4 μM and 5.3 ± 1.5 μM, 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 mc226 strain of M. smegmatis.

Original languageEnglish (US)
Pages (from-to)184-190
Number of pages7
JournalExperimental Parasitology
Volume72
Issue number2
DOIs
StatePublished - Feb 1991
Externally publishedYes

Keywords

  • Dihydrofolate reductase
  • Mutation
  • Mycobacterium smegmatis
  • Trimethoprim

ASJC Scopus subject areas

  • Parasitology
  • Immunology
  • Infectious Diseases

Fingerprint Dive into the research topics of 'Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis'. Together they form a unique fingerprint.

  • Cite this