Résumé des résultats (Abstract)
(Anglais)
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The objective of this program was to obtain molecular insight into the structure and function of the catalase-peroxidase KatG and alkylhydroperoxidase AhpC; to elucidate their precise roles in the mechanism of action of isoniazid (INH), in INH resistance and in mycobacterial virulence; and to identify and characterize compounds that interact with these enzymes and could ultimately serve as new antituberculosis drugs.
In order to understand the mechanism of INH, our laboratory first studied the transformation of INH, which is a pro-drug, into its bioactive form by the catalase-peroxidase KatG. We were able to elucidate the reaction mechanism leading to the formation of an INH-nicotinamide adenine dinucleotide adduct (INH-NAD). It is the INH-NAD adduct that inhibits the synthesis of mycolic acids in M. tuberculosis by binding to and inhibiting the enoyl acyl carrier protein reductase InhA. In contrast to earlier speculations, this adduct is formed by a Minisci-reaction after the oxidation of INH to an isonicotinoyl radical by KatG. We were also able to synthesize the INH-NAD adduct and measure its inhibitory activity against InhA for the first time. The proposed mechanism of action of INH has important implications for our understanding of INH sensitivity and resistance in mycobacteria. This work is described in the following publication: M. Wilming, K. Johnsson Angew. Chem. Inter. Ed. 38, 2588-2590 (1999).
In collaboration with the laboratories of S. Cole and P. Alzari we also investigated a mutant of the catalase-peroxidase KatG that has been isolated out of INH-resistant clinical M. tuberculosis strains (Ser315Thr). We were able to show that this mutation leads to a loss in peroxidase activity, resulting in a lower rate of formation of both the isonicotinoyl radical of INH as well as the INH-NAD adduct and, consequently, to INH resistance. This work is described in the following publication: Saint-Joanis B, Souchon H, Wilming M, Johnsson K, Alzari PM, Cole ST Biochem J 1999, 338, 753-60.
To study the structure-function relationship of class I peroxidases such as KatG as well as the homologous cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae, we subjected CCP to directed molecular evolution to generate mutants with increased activity against the classical peroxidase substrate guaiacol, thus changing the substrate specificity of CCP from the protein cytochrome c to a small organic molecule. Mutants were isolated which significantly increased activities against small organic substrates and allowed to identify residues that play a decisive role in controlling substrate specificity in peroxidases. This work is described in the following publication: Iffland A, Tafelmeyer P, Saudan C, Johnsson K Biochemistry 2000, 39, 10790-8.
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