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

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

Fingerprint

Molecular Sequence Data
Trimethoprim Resistance
Mycobacterium smegmatis
Tetrahydrofolate Dehydrogenase
Trimethoprim
Mycobacterium
Affinity Chromatography
Polyacrylamide Gel Electrophoresis
Amino Acid Sequence
Mutation
Enzymes
NADP
Pharmaceutical Preparations
Pyrimethamine
Complex Mixtures
Sepharose
Gel Chromatography
Sequence Analysis

Keywords

  • Dihydrofolate reductase
  • Mutation
  • Mycobacterium smegmatis
  • Trimethoprim

ASJC Scopus subject areas

  • Parasitology
  • Immunology
  • Infectious Diseases

Cite this

Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis. / Sirawaraporn, Worachart; Sirawaraporn, Rachada; Chanpongsri, Atid; Jacobs, William R.; Santi, Daniel V.

In: Experimental Parasitology, Vol. 72, No. 2, 02.1991, p. 184-190.

Research output: Contribution to journalArticle

Sirawaraporn, Worachart ; Sirawaraporn, Rachada ; Chanpongsri, Atid ; Jacobs, William R. ; Santi, Daniel V. / Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis. In: Experimental Parasitology. 1991 ; Vol. 72, No. 2. pp. 184-190.
@article{96ce388fb10042ec99c3acff626e57ef,
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 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.",
keywords = "Dihydrofolate reductase, Mutation, Mycobacterium smegmatis, Trimethoprim",
author = "Worachart Sirawaraporn and Rachada Sirawaraporn and Atid Chanpongsri and Jacobs, {William R.} and Santi, {Daniel V.}",
year = "1991",
month = "2",
doi = "10.1016/0014-4894(91)90136-K",
language = "English (US)",
volume = "72",
pages = "184--190",
journal = "Experimental Parasitology",
issn = "0014-4894",
publisher = "Academic Press Inc.",
number = "2",

}

TY - JOUR

T1 - Purification and characterization of dihydrofolate reductase from wild-type and trimethoprim-resistant Mycobacterium smegmatis

AU - Sirawaraporn, Worachart

AU - Sirawaraporn, Rachada

AU - Chanpongsri, Atid

AU - Jacobs, William R.

AU - Santi, Daniel V.

PY - 1991/2

Y1 - 1991/2

N2 - 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.

AB - 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.

KW - Dihydrofolate reductase

KW - Mutation

KW - Mycobacterium smegmatis

KW - Trimethoprim

UR - http://www.scopus.com/inward/record.url?scp=0026113224&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026113224&partnerID=8YFLogxK

U2 - 10.1016/0014-4894(91)90136-K

DO - 10.1016/0014-4894(91)90136-K

M3 - Article

C2 - 2009922

AN - SCOPUS:0026113224

VL - 72

SP - 184

EP - 190

JO - Experimental Parasitology

JF - Experimental Parasitology

SN - 0014-4894

IS - 2

ER -