In vitro inhibition of the Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein reductase MabA by isoniazid.
The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent beta-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between Mn(III)-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP(+). The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the beta-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.
Ducasse-Cabanot
Stéphanie
S
Institut de Pharmacologie et de Biologie Structurale, Département des Mécanismes Moléculaires des Infections Mycobactériennes, CNRS, Toulouse, France.
Cohen-Gonsaud
Martin
M
Marrakchi
Hedia
H
Nguyen
Michel
M
Zerbib
Didier
D
Bernadou
Jean
J
Daffé
Mamadou
M
Labesse
Gilles
G
Quémard
Annaïk
A
D000213:Acyl Carrier Protein / Q000235:genetics / Q000378:metabolism; D000995:Antitubercular Agents / Q000494:pharmacology*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D018360:Crystallography, X-Ray; D005227:Fatty Acids / Q000378:metabolism; D007538:Isoniazid / Q000494:pharmacology*; D008958:Models, Molecular; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000201:enzymology* / Q000235:genetics; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D021241:Spectrometry, Mass, Electrospray Ionization
D000213:Acyl Carrier Protein; D000995:Antitubercular Agents; D005227:Fatty Acids; D009249:NADP; D007538:Isoniazid