Nadh binding to wild-type and mutant forms of inha from isoniazid-resistant clinical isolates of mycobacterium tuberculosis

Luiz A. Basso, Renjian Zheng, James M. Musser, John S. Blanchard

Research output: Contribution to journalArticle

Abstract

The Mycobacterium tuberculosis inhA-eneoded enoyl reductase has been proposed as the target of isoniazid, the most widely prescribed antitubercular drug. Isoniazid-dependent inactivation of the enoyl reductase has been suggested to require interaction of an activated form of the drug with enzymebound NADH. We have determined the binding of NADH to the wildtype enzyme using fluorescence titration, since the fluorescence of NADH is significantly enhanced upon binding to the enzyme. NADH binds tightly (Kd=0. 6μM) and stoichiometrically to the enzyme, and exhibits hyperbolic saturation. The S94A mutant has been previously identified from isoniazidresistant laboratory strains of M. smegmatis, and the I16T and V78A mutants have been identified from isoniazid-resistant clinical isolates of M. tuberculosis. These three mutants were prepared, expressed in E. coli and purified to homogeneity. All three mutants exhibited significantly lower affinities for NADH (Kd values ranged from 6-36#M), and displayed sigmoidal binding curves. These data support our proposal that inactivation of the enoyl reductase requires interaction of the activated drug with enzyme-bound NADH, and that isoniasid resistance can be accounted for by mutations which lead to decreases in the affinity for NADH.

Original languageEnglish (US)
JournalFASEB Journal
Volume11
Issue number9
StatePublished - 1997

Fingerprint

isoniazid
Isoniazid
Mycobacterium tuberculosis
NAD
mutants
enzymes
drugs
inactivation
fluorescence
Oxidoreductases
Enzymes
titration
Smegma
Fluorescence
Antitubercular Agents
Escherichia coli
mutation
Titration
Drug Interactions
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Nadh binding to wild-type and mutant forms of inha from isoniazid-resistant clinical isolates of mycobacterium tuberculosis. / Basso, Luiz A.; Zheng, Renjian; Musser, James M.; Blanchard, John S.

In: FASEB Journal, Vol. 11, No. 9, 1997.

Research output: Contribution to journalArticle

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AU - Blanchard, John S.

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AB - The Mycobacterium tuberculosis inhA-eneoded enoyl reductase has been proposed as the target of isoniazid, the most widely prescribed antitubercular drug. Isoniazid-dependent inactivation of the enoyl reductase has been suggested to require interaction of an activated form of the drug with enzymebound NADH. We have determined the binding of NADH to the wildtype enzyme using fluorescence titration, since the fluorescence of NADH is significantly enhanced upon binding to the enzyme. NADH binds tightly (Kd=0. 6μM) and stoichiometrically to the enzyme, and exhibits hyperbolic saturation. The S94A mutant has been previously identified from isoniazidresistant laboratory strains of M. smegmatis, and the I16T and V78A mutants have been identified from isoniazid-resistant clinical isolates of M. tuberculosis. These three mutants were prepared, expressed in E. coli and purified to homogeneity. All three mutants exhibited significantly lower affinities for NADH (Kd values ranged from 6-36#M), and displayed sigmoidal binding curves. These data support our proposal that inactivation of the enoyl reductase requires interaction of the activated drug with enzyme-bound NADH, and that isoniasid resistance can be accounted for by mutations which lead to decreases in the affinity for NADH.

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