Action Mechanism of Antitubercular Isoniazid

Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an inhibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the inhibitor has never been isolated. We have purified the InhA-inhibitor complex generated in the M. tuberculosis KatG-catalyzed INH activation. The complex exhibited a 278-nm absorption peak and a shoulder around 326 nm with a characteristicA 326/A 278 ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a K d < 0.4 nm and can be dissociated from denatured InhA for chromatographic isolation. The free inhibitor showed absorption peaks at 326 (ε326 6900 m−1cm−1) and 260 nm (ε260 27,000m−1 cm−1). The inactive complex can be reconstituted from InhA and the isolated inhibitor. The InhA inhibitor from the KatG-catalyzed INH activation was identical to that from a slow, KatG-independent, Mn2+-mediated reaction based on high pressure liquid chromatography analysis and absorption and mass spectral characteristics. By monitoring the formation of the InhA-inhibitor complex, we have found that manganese is not essential to the INH activation by M. tuberculosis KatG. Furthermore, the formation of the InhA inhibitor in the KatG reaction was independent of InhA. Activation of the antitubercular isoniazid (INH) by the Mycobacterium tuberculosis KatG produces an inhibitor for enoyl reductase (InhA). The mechanism for INH activation remains poorly understood, and the inhibitor has never been isolated. We have purified the InhA-inhibitor complex generated in the M. tuberculosis KatG-catalyzed INH activation. The complex exhibited a 278-nm absorption peak and a shoulder around 326 nm with a characteristicA 326/A 278 ratio of 0.16. The complex was devoid of enoyl reductase activity. The inhibitor noncovalently binds to InhA with a K d < 0.4 nm and can be dissociated from denatured InhA for chromatographic isolation. The free inhibitor showed absorption peaks at 326 (ε326 6900 m−1cm−1) and 260 nm (ε260 27,000m−1 cm−1). The inactive complex can be reconstituted from InhA and the isolated inhibitor. The InhA inhibitor from the KatG-catalyzed INH activation was identical to that from a slow, KatG-independent, Mn2+-mediated reaction based on high pressure liquid chromatography analysis and absorption and mass spectral characteristics. By monitoring the formation of the InhA-inhibitor complex, we have found that manganese is not essential to the INH activation by M. tuberculosis KatG. Furthermore, the formation of the InhA inhibitor in the KatG reaction was independent of InhA. isoniazid or isonicotinic acid hydrazide enoyl-acyl carrier protein reductase high pressure liquid chromatography isonicotinic acid coenzyme A Tuberculosis due to Mycobacterium tuberculosisinfection is the leading cause of death worldwide among known infectious diseases. A sizeable increase of tuberculosis cases in the United States since 1985 (1.Bloom B.R. Murray C.J.L. Science. 1992; 257: 1055-1064Crossref PubMed Scopus (1236) Google Scholar) is followed by a decrease in more recent years. However, ∼13% of recent cases involve M. tuberculosis strains that are resistant to one or more frontline antitubercular drugs such as isoniazid (isonicotinic acid hydrazide, INH),1 rifampicin, and streptomycin (2.Frieden T.R. Sterling T. Pablos-Mendez A. Kilburn J.O. Cauthen G.M. Dooley S.W. N. Engl. J. Med. 1993; 328: 521-526Crossref PubMed Scopus (849) Google Scholar, 3.Bloch A.B. Cauthen G.M. Onorato I.M. Dansbury K.G. Kelly G.D. Driver C.R. Snider D.E. J. Am. Med. Assoc. 1994; 271: 665-671Crossref PubMed Scopus (335) Google Scholar, 4.Bifani P. Plikaytis B.B. Kapur V. Stockbauer K. Pan X. Lutfey M.L. Moghazeh S.L. Eisner W. Daniel T.M. Kaplan M.H. Crawford J.T. Musser J.M. Kreiswirth B.N. J. Am. Med. Assoc. 1996; 275: 452-457Crossref PubMed Google Scholar).INH has been the cornerstone in tuberculosis chemotherapy for almost half a century since its discovery as a potent antituberculosis drug in 1952 (5.Bernstein J. Lott W.A. Steinberg B.A. Yale H.L. Am. Rev. Tuber. 1952; 65: 357-364PubMed Google Scholar, 6.Fox H.H. Science. 1952; 116: 129-134Crossref PubMed Scopus (90) Google Scholar, 7.Pansy F. Stander H. Donovick R. Am. Rev. Tuber. 1952; 65: 761-764PubMed Google Scholar). INH is a prodrug, and its antituberculosis function requires in vivo activation by KatG, an enzyme with dual activities of catalase and peroxidase. The involvement of KatG in the INH action was first implied by an apparent correlation between the loss of KatG catalase activity and INH resistance (8.Middlebrook G. Cohn M.L. Schaefer W.B. Am. Rev. Tuberc. 1954; 70: 852-872Crossref PubMed Google Scholar) and confirmed by a genetic study (9.Zhang Y. Heym B. Allen B. Young D. Stewart C. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1086) Google Scholar). Clinical M. tuberculosis isolates resistant to INH were subsequently revealed to have various alterations in the katG gene (10.Altamirano M. Marostenmaki J. Wong A. FitzGerald M. Black W.A. Smith J.A. J. Infect. Dis. 1994; 169: 162-165Crossref PubMed Scopus (55) Google Scholar, 11.Heym B. Alzari P.M. Honoré N. Cole S.T. Mol. Microbiol. 1995; 15: 235-245Crossref PubMed Scopus (309) Google Scholar, 12.Musser J.M. Kapur V. Williams D.L. Kreiswirth B.N. van Soolingen D. van Embden J.D.A. J. Infect. Dis. 1996; 173: 196-202Crossref PubMed Scopus (299) Google Scholar). INH activation leads to inhibition of the synthesis of mycolic acid, a long chain fatty acid-containing component of the mycobacterial cell wall (13.Winder F.G. Collins P.B. J. Gen. Microbiol. 1970; 63: 41-48Crossref PubMed Scopus (173) Google Scholar, 14.Takayama K. Wang L. David H.L. Antimicrob. Agents Chemother. 1972; 2: 29-35Crossref PubMed Scopus (228) Google Scholar). Two enzymes involved in the elongation cycle of the fatty acid biosynthesis, namely an enoyl-acyl carrier protein reductase (InhA) (15.Banerjee A. Dubnau E. Quemard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar, 16.Quémard A. Sacchettini J.C. Dessen A. Vilcheze C. Bittman R. Jacobs W.R. Blanchard J.S. Biochemistry. 1995; 34: 8235-8241Crossref PubMed Scopus (327) Google Scholar) and β-ketoacyl-acyl carrier protein synthase (17.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar), are believed to be targets of the activated inhibitor(s).Progress made thus far notwithstanding the mechanisms of INH action and resistance are still poorly understood. Purified KatG from eitherM. tuberculosis (18.Johnsson K. King D.S. Schultz P.G. J. Am. Chem. Soc. 1995; 117: 5009-5010Crossref Scopus (202) Google Scholar) or Mycobacterium smegmatis(19.Zabinski R.F. Blanchard J.S. J. Am. Chem. Soc. 1997; 119: 2331-2332Crossref Scopus (59) Google Scholar) catalyzes the in vitro inactivation of InhA by INH in the presence of NADH and Mn2+. However, the molecular nature for the INH activation by KatG and the functional role of Mn2+ remain unclear. InhA is also inactivated in a slow nonenzymatic, Mn2+-dependent activation of INH. The crystal structure of the resulting InhA-inhibitor complex has been determined, which shows that the bound inhibitor is an isonicotinic acyl NADH (20.Rozwarski D. Grant G.A. Barton D.H.R. Jacobs W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (605) Google Scholar). It is, however, uncertain whether or not the inhibitor generated by this nonenzymatic activation is identical to that formed in the KatG-dependent process. Moreover, the inhibitor derived from INH by either the nonenzymatic or the KatG-dependent activation has never been isolated, and no simple method has been developed for the detection and quantification of the inhibitor. Consequently, biochemical or biophysical characterizations of the nature and consequences of the inhibitor binding by InhA have been greatly hindered by these limitations.We are interested in the mechanisms of the INH action and resistance. This work was carried out to isolate for the first time the InhA inhibitors generated by the nonenzymatic and the M. tuberculosis KatG-dependent processes and to characterize the free inhibitors and the InhA-inhibitor complexes. Evidence is also presented to show that neither Mn2+ nor InhA is essential to the M. tuberculosis KatG-mediated activation of INH.DISCUSSIONAn InhA-inhibitor complex can be obtained by a slow INH activation reaction that is Mn2+-mediated but KatG-independent; the identity of the bound inhibitor as isonicotinic acyl NADH has been established from the determination of the crystal structure of such a complex (20.Rozwarski D. Grant G.A. Barton D.H.R. Jacobs W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (605) Google Scholar). An InhA inhibitor can also be obtained by a rapid KatG-dependent activation of INH. However, it is not clear whether these two inhibitors are the same. Moreover, the inhibitor derived from either activation process has never been isolated in solution. In this work, procedures were developed for the isolation and quantification of the InhA-inhibitor complex and the free inhibitor using either the M. tuberculosis KatG-dependent or the Mn2+-mediated process for the INH activation. The InhA-inhibitor complex obtained by the KatG-dependent activation of INH was apparently identical to its counterpart derived from the Mn2+-mediated activation with respect to absorption spectra and the lack of enoyl reductase activity. The free inhibitors obtained from these two complexes were also identical in their absorption spectra, HPLC retention times, and mass spectra. These results indicate that the same InhA inhibitor was generated by either the slow Mn2+-mediated or the fast KatG-catalyzed INH activation.Both the free and the InhA-bound inhibitor were sufficiently stable to allow the isolation and the subsequent characterization of the inhibitor. The complexes derived from both activation processes were indistinguishable from those reconstituted from InhA and the isolated free inhibitor with respect to absorption spectra. The binding of the inhibitor to InhA was apparently very tight with a dissociation constant estimated to be lower than 0.4 nm. The free inhibitor has absorption peaks at 260 and 326 nm (Fig. 2 A). In comparison with NADH, the 326-nm peak of the inhibitor is stronger in absorptivity (ε326 about 6900m−1 cm−1) and in a shorter wavelength range than the NADH 340-nm peak (ε340 6200m−1 cm−1). Upon binding to InhA, the inhibitor 326-nm peak was blue-shifted, but the complex still showed a pronounced shoulder around 320 nm (Fig. 2 A). The characteristic absorption around 320 nm provides a very useful reporting signal for monitoring the formation and isolation of the inhibitor. The spectral changes resulting from the inhibitor binding by InhA, especially the ΔA 353 (Fig.2 B), also provide a convenient means for investigating the binding of the inhibitor by InhA. Using this method, the KatG S315T mutant frequently encountered in INH-resistant M. tuberculosis isolates has been shown to fail to catalyze the formation of the InhA inhibitor. 2B. Lei and S.-C. Tu, unpublished results. We believe that the methodology developed in this report can also be applied to a recently identified INH target KasA (17.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar) for further verification and investigation of the inactivation mechanism.There are some debates about whether InhA is a primary target of activated INH in M. tuberculosis (29.Mdluli K. Sherman D.R. Hickey M.J. Kreiswirth B.N. Morris S. Stover C.K. Barry III, C.E. J. Infect. Dis. 1996; 174: 1085-1090Crossref PubMed Scopus (102) Google Scholar), in part due to the infrequency of mutation in the inhA gene in INH-resistantM. tuberculosis strains. Particularly, the S92A mutation of InhA, which confers high INH resistance in M. smegmatis(15.Banerjee A. Dubnau E. Quemard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar), has not been encountered in M. tuberculosis isolates from INH-resistant patients. Our findings support InhA as a primary target of INH action in M. tuberculosis. KatG catalyzes the formation of the InhA inhibitor. The extremely high affinity of the inhibitor to InhA (K d < 0.4 nm) is consistent with the high susceptibility of M. tuberculosisto INH. There are two possibilities for the lack of the S92A InhA allele in M. tuberculosis. One is that the selection pressure is not high enough for the mutation. M. smegmatis(which does not cause tuberculosis) and M. tuberculosis are sensitive to INH with minimum inhibitory concentrations of > 5 and 0.01–0.02 μg/ml, respectively. The M. smegmatis InhA S92A mutant confers INH resistance of minimum inhibitory concentrations > 50 μg/ml, whereas most of clinical INH-resistant M. tuberculosis isolates have minimum inhibitory concentrations of 1–5 μg/ml. In clinical treatments, INH was absorbed by the gut to reach peak levels of 3–7 μg/ml in 1–2 h after a usual oral dosage of 300 mg (30.Holdiness M.R. Clin. Pharmacokinet. 1984; 9: 511-544Crossref PubMed Scopus (194) Google Scholar). At such an in vivolevel of INH, the InhA S92A mutation of M. tuberculosiswould not be effectively selected. The other possibility is that, because the InhA enzymes from M. tuberculosis and M. smegmatis have 11.7% nonidentity in their amino acid sequences, the M. tuberculosis InhA S92A mutant may be distinct from the corresponding M. smegmatis mutant in remaining sensitive to the inhibitor. This latter possibility is under current investigation.The M. smegmatis KatG has been shown to require Mn2+ for the activation of INH for the inhibition of InhA (19.Zabinski R.F. Blanchard J.S. J. Am. Chem. Soc. 1997; 119: 2331-2332Crossref Scopus (59) Google Scholar), possibly by converting Mn2+ to Mn3+, which in turn oxidizes INH (31.Magliozzo R.S. Marcinkeviciene J.A. J. Biol. Chem. 1997; 272: 8867-8870Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We found that, although Mn2+ enhanced the INH activation by M. tuberculosis KatG, the M. tuberculosis KatG can efficiently activate INH without exogenously added Mn2+. We did not find any detectable amount of Mn2+ in the Pi buffer, the purified InhA, or the purified KatG used in the activation reaction. Therefore, Mn2+ is apparently not essential to the activation of INH by M. tuberculosis KatG. The two KatG enzymes from M. tuberculosis and M. smegmatis are thus different in their modes of the INH activation. Such a difference might be related to the differential susceptibilities of M. tuberculosis and M. smegmatis to INH.Previous in vitro experiments of INH activation all included InhA in the reaction. It is not clear whether InhA is required in addition to KatG for INH activation. A similar level of the InhA inhibitor was generated in the KatG reactions with or without InhA. Therefore, the simultaneous presence of InhA and KatG is not required for the inhibitor production.KatG used in this report had a Met-Asn-Ser tripeptide fused to the first residue Met of the wild type KatG. In comparison with the wild-type KatG, the purified modified enzyme exhibited essentially the same A 408/A 280 ratio, kinetic parameters, the ability to activate INH, and blue shift of the Soret band A 408 (32.Wengenack N.L. Todorovic S., Yu, L. Rusnak F. Biochemistry. 1998; 37: 15825-15834Crossref PubMed Scopus (70) Google Scholar) upon INH binding (not shown). The extra peptide apparently does not significantly change the structure or function of KatG. This is in contrast with another fusion enzyme that had a Met-Glu-Phe-Val tetrapeptide fused to the second residue Pro. This latter modified KatG bound only about 0.5 heme/dimer, in comparison with 2 heme/dimer by the wild type and our modified KatG, and thus had a much lower enzyme activity (33.Nagy J.M. Cass A.E.G. Brown K.A. J. Biol. Chem. 1997; 272: 31265-31271Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A constitutive expression was adopted to slowly accumulate our enzyme while the other KatG fusion was overexpressed with an inducible system. The difference in the expression strategy could lead to the difference in the incorporation of the heme cofactor. Tuberculosis due to Mycobacterium tuberculosisinfection is the leading cause of death worldwide among known infectious diseases. A sizeable increase of tuberculosis cases in the United States since 1985 (1.Bloom B.R. Murray C.J.L. Science. 1992; 257: 1055-1064Crossref PubMed Scopus (1236) Google Scholar) is followed by a decrease in more recent years. However, ∼13% of recent cases involve M. tuberculosis strains that are resistant to one or more frontline antitubercular drugs such as isoniazid (isonicotinic acid hydrazide, INH),1 rifampicin, and streptomycin (2.Frieden T.R. Sterling T. Pablos-Mendez A. Kilburn J.O. Cauthen G.M. Dooley S.W. N. Engl. J. Med. 1993; 328: 521-526Crossref PubMed Scopus (849) Google Scholar, 3.Bloch A.B. Cauthen G.M. Onorato I.M. Dansbury K.G. Kelly G.D. Driver C.R. Snider D.E. J. Am. Med. Assoc. 1994; 271: 665-671Crossref PubMed Scopus (335) Google Scholar, 4.Bifani P. Plikaytis B.B. Kapur V. Stockbauer K. Pan X. Lutfey M.L. Moghazeh S.L. Eisner W. Daniel T.M. Kaplan M.H. Crawford J.T. Musser J.M. Kreiswirth B.N. J. Am. Med. Assoc. 1996; 275: 452-457Crossref PubMed Google Scholar). INH has been the cornerstone in tuberculosis chemotherapy for almost half a century since its discovery as a potent antituberculosis drug in 1952 (5.Bernstein J. Lott W.A. Steinberg B.A. Yale H.L. Am. Rev. Tuber. 1952; 65: 357-364PubMed Google Scholar, 6.Fox H.H. Science. 1952; 116: 129-134Crossref PubMed Scopus (90) Google Scholar, 7.Pansy F. Stander H. Donovick R. Am. Rev. Tuber. 1952; 65: 761-764PubMed Google Scholar). INH is a prodrug, and its antituberculosis function requires in vivo activation by KatG, an enzyme with dual activities of catalase and peroxidase. The involvement of KatG in the INH action was first implied by an apparent correlation between the loss of KatG catalase activity and INH resistance (8.Middlebrook G. Cohn M.L. Schaefer W.B. Am. Rev. Tuberc. 1954; 70: 852-872Crossref PubMed Google Scholar) and confirmed by a genetic study (9.Zhang Y. Heym B. Allen B. Young D. Stewart C. Nature. 1992; 358: 591-593Crossref PubMed Scopus (1086) Google Scholar). Clinical M. tuberculosis isolates resistant to INH were subsequently revealed to have various alterations in the katG gene (10.Altamirano M. Marostenmaki J. Wong A. FitzGerald M. Black W.A. Smith J.A. J. Infect. Dis. 1994; 169: 162-165Crossref PubMed Scopus (55) Google Scholar, 11.Heym B. Alzari P.M. Honoré N. Cole S.T. Mol. Microbiol. 1995; 15: 235-245Crossref PubMed Scopus (309) Google Scholar, 12.Musser J.M. Kapur V. Williams D.L. Kreiswirth B.N. van Soolingen D. van Embden J.D.A. J. Infect. Dis. 1996; 173: 196-202Crossref PubMed Scopus (299) Google Scholar). INH activation leads to inhibition of the synthesis of mycolic acid, a long chain fatty acid-containing component of the mycobacterial cell wall (13.Winder F.G. Collins P.B. J. Gen. Microbiol. 1970; 63: 41-48Crossref PubMed Scopus (173) Google Scholar, 14.Takayama K. Wang L. David H.L. Antimicrob. Agents Chemother. 1972; 2: 29-35Crossref PubMed Scopus (228) Google Scholar). Two enzymes involved in the elongation cycle of the fatty acid biosynthesis, namely an enoyl-acyl carrier protein reductase (InhA) (15.Banerjee A. Dubnau E. Quemard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar, 16.Quémard A. Sacchettini J.C. Dessen A. Vilcheze C. Bittman R. Jacobs W.R. Blanchard J.S. Biochemistry. 1995; 34: 8235-8241Crossref PubMed Scopus (327) Google Scholar) and β-ketoacyl-acyl carrier protein synthase (17.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar), are believed to be targets of the activated inhibitor(s). Progress made thus far notwithstanding the mechanisms of INH action and resistance are still poorly understood. Purified KatG from eitherM. tuberculosis (18.Johnsson K. King D.S. Schultz P.G. J. Am. Chem. Soc. 1995; 117: 5009-5010Crossref Scopus (202) Google Scholar) or Mycobacterium smegmatis(19.Zabinski R.F. Blanchard J.S. J. Am. Chem. Soc. 1997; 119: 2331-2332Crossref Scopus (59) Google Scholar) catalyzes the in vitro inactivation of InhA by INH in the presence of NADH and Mn2+. However, the molecular nature for the INH activation by KatG and the functional role of Mn2+ remain unclear. InhA is also inactivated in a slow nonenzymatic, Mn2+-dependent activation of INH. The crystal structure of the resulting InhA-inhibitor complex has been determined, which shows that the bound inhibitor is an isonicotinic acyl NADH (20.Rozwarski D. Grant G.A. Barton D.H.R. Jacobs W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (605) Google Scholar). It is, however, uncertain whether or not the inhibitor generated by this nonenzymatic activation is identical to that formed in the KatG-dependent process. Moreover, the inhibitor derived from INH by either the nonenzymatic or the KatG-dependent activation has never been isolated, and no simple method has been developed for the detection and quantification of the inhibitor. Consequently, biochemical or biophysical characterizations of the nature and consequences of the inhibitor binding by InhA have been greatly hindered by these limitations. We are interested in the mechanisms of the INH action and resistance. This work was carried out to isolate for the first time the InhA inhibitors generated by the nonenzymatic and the M. tuberculosis KatG-dependent processes and to characterize the free inhibitors and the InhA-inhibitor complexes. Evidence is also presented to show that neither Mn2+ nor InhA is essential to the M. tuberculosis KatG-mediated activation of INH. DISCUSSIONAn InhA-inhibitor complex can be obtained by a slow INH activation reaction that is Mn2+-mediated but KatG-independent; the identity of the bound inhibitor as isonicotinic acyl NADH has been established from the determination of the crystal structure of such a complex (20.Rozwarski D. Grant G.A. Barton D.H.R. Jacobs W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (605) Google Scholar). An InhA inhibitor can also be obtained by a rapid KatG-dependent activation of INH. However, it is not clear whether these two inhibitors are the same. Moreover, the inhibitor derived from either activation process has never been isolated in solution. In this work, procedures were developed for the isolation and quantification of the InhA-inhibitor complex and the free inhibitor using either the M. tuberculosis KatG-dependent or the Mn2+-mediated process for the INH activation. The InhA-inhibitor complex obtained by the KatG-dependent activation of INH was apparently identical to its counterpart derived from the Mn2+-mediated activation with respect to absorption spectra and the lack of enoyl reductase activity. The free inhibitors obtained from these two complexes were also identical in their absorption spectra, HPLC retention times, and mass spectra. These results indicate that the same InhA inhibitor was generated by either the slow Mn2+-mediated or the fast KatG-catalyzed INH activation.Both the free and the InhA-bound inhibitor were sufficiently stable to allow the isolation and the subsequent characterization of the inhibitor. The complexes derived from both activation processes were indistinguishable from those reconstituted from InhA and the isolated free inhibitor with respect to absorption spectra. The binding of the inhibitor to InhA was apparently very tight with a dissociation constant estimated to be lower than 0.4 nm. The free inhibitor has absorption peaks at 260 and 326 nm (Fig. 2 A). In comparison with NADH, the 326-nm peak of the inhibitor is stronger in absorptivity (ε326 about 6900m−1 cm−1) and in a shorter wavelength range than the NADH 340-nm peak (ε340 6200m−1 cm−1). Upon binding to InhA, the inhibitor 326-nm peak was blue-shifted, but the complex still showed a pronounced shoulder around 320 nm (Fig. 2 A). The characteristic absorption around 320 nm provides a very useful reporting signal for monitoring the formation and isolation of the inhibitor. The spectral changes resulting from the inhibitor binding by InhA, especially the ΔA 353 (Fig.2 B), also provide a convenient means for investigating the binding of the inhibitor by InhA. Using this method, the KatG S315T mutant frequently encountered in INH-resistant M. tuberculosis isolates has been shown to fail to catalyze the formation of the InhA inhibitor. 2B. Lei and S.-C. Tu, unpublished results. We believe that the methodology developed in this report can also be applied to a recently identified INH target KasA (17.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar) for further verification and investigation of the inactivation mechanism.There are some debates about whether InhA is a primary target of activated INH in M. tuberculosis (29.Mdluli K. Sherman D.R. Hickey M.J. Kreiswirth B.N. Morris S. Stover C.K. Barry III, C.E. J. Infect. Dis. 1996; 174: 1085-1090Crossref PubMed Scopus (102) Google Scholar), in part due to the infrequency of mutation in the inhA gene in INH-resistantM. tuberculosis strains. Particularly, the S92A mutation of InhA, which confers high INH resistance in M. smegmatis(15.Banerjee A. Dubnau E. Quemard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar), has not been encountered in M. tuberculosis isolates from INH-resistant patients. Our findings support InhA as a primary target of INH action in M. tuberculosis. KatG catalyzes the formation of the InhA inhibitor. The extremely high affinity of the inhibitor to InhA (K d < 0.4 nm) is consistent with the high susceptibility of M. tuberculosisto INH. There are two possibilities for the lack of the S92A InhA allele in M. tuberculosis. One is that the selection pressure is not high enough for the mutation. M. smegmatis(which does not cause tuberculosis) and M. tuberculosis are sensitive to INH with minimum inhibitory concentrations of > 5 and 0.01–0.02 μg/ml, respectively. The M. smegmatis InhA S92A mutant confers INH resistance of minimum inhibitory concentrations > 50 μg/ml, whereas most of clinical INH-resistant M. tuberculosis isolates have minimum inhibitory concentrations of 1–5 μg/ml. In clinical treatments, INH was absorbed by the gut to reach peak levels of 3–7 μg/ml in 1–2 h after a usual oral dosage of 300 mg (30.Holdiness M.R. Clin. Pharmacokinet. 1984; 9: 511-544Crossref PubMed Scopus (194) Google Scholar). At such an in vivolevel of INH, the InhA S92A mutation of M. tuberculosiswould not be effectively selected. The other possibility is that, because the InhA enzymes from M. tuberculosis and M. smegmatis have 11.7% nonidentity in their amino acid sequences, the M. tuberculosis InhA S92A mutant may be distinct from the corresponding M. smegmatis mutant in remaining sensitive to the inhibitor. This latter possibility is under current investigation.The M. smegmatis KatG has been shown to require Mn2+ for the activation of INH for the inhibition of InhA (19.Zabinski R.F. Blanchard J.S. J. Am. Chem. Soc. 1997; 119: 2331-2332Crossref Scopus (59) Google Scholar), possibly by converting Mn2+ to Mn3+, which in turn oxidizes INH (31.Magliozzo R.S. Marcinkeviciene J.A. J. Biol. Chem. 1997; 272: 8867-8870Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We found that, although Mn2+ enhanced the INH activation by M. tuberculosis KatG, the M. tuberculosis KatG can efficiently activate INH without exogenously added Mn2+. We did not find any detectable amount of Mn2+ in the Pi buffer, the purified InhA, or the purified KatG used in the activation reaction. Therefore, Mn2+ is apparently not essential to the activation of INH by M. tuberculosis KatG. The two KatG enzymes from M. tuberculosis and M. smegmatis are thus different in their modes of the INH activation. Such a difference might be related to the differential susceptibilities of M. tuberculosis and M. smegmatis to INH.Previous in vitro experiments of INH activation all included InhA in the reaction. It is not clear whether InhA is required in addition to KatG for INH activation. A similar level of the InhA inhibitor was generated in the KatG reactions with or without InhA. Therefore, the simultaneous presence of InhA and KatG is not required for the inhibitor production.KatG used in this report had a Met-Asn-Ser tripeptide fused to the first residue Met of the wild type KatG. In comparison with the wild-type KatG, the purified modified enzyme exhibited essentially the same A 408/A 280 ratio, kinetic parameters, the ability to activate INH, and blue shift of the Soret band A 408 (32.Wengenack N.L. Todorovic S., Yu, L. Rusnak F. Biochemistry. 1998; 37: 15825-15834Crossref PubMed Scopus (70) Google Scholar) upon INH binding (not shown). The extra peptide apparently does not significantly change the structure or function of KatG. This is in contrast with another fusion enzyme that had a Met-Glu-Phe-Val tetrapeptide fused to the second residue Pro. This latter modified KatG bound only about 0.5 heme/dimer, in comparison with 2 heme/dimer by the wild type and our modified KatG, and thus had a much lower enzyme activity (33.Nagy J.M. Cass A.E.G. Brown K.A. J. Biol. Chem. 1997; 272: 31265-31271Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A constitutive expression was adopted to slowly accumulate our enzyme while the other KatG fusion was overexpressed with an inducible system. The difference in the expression strategy could lead to the difference in the incorporation of the heme cofactor. An InhA-inhibitor complex can be obtained by a slow INH activation reaction that is Mn2+-mediated but KatG-independent; the identity of the bound inhibitor as isonicotinic acyl NADH has been established from the determination of the crystal structure of such a complex (20.Rozwarski D. Grant G.A. Barton D.H.R. Jacobs W.R. Sacchettini J.C. Science. 1998; 279: 98-102Crossref PubMed Scopus (605) Google Scholar). An InhA inhibitor can also be obtained by a rapid KatG-dependent activation of INH. However, it is not clear whether these two inhibitors are the same. Moreover, the inhibitor derived from either activation process has never been isolated in solution. In this work, procedures were developed for the isolation and quantification of the InhA-inhibitor complex and the free inhibitor using either the M. tuberculosis KatG-dependent or the Mn2+-mediated process for the INH activation. The InhA-inhibitor complex obtained by the KatG-dependent activation of INH was apparently identical to its counterpart derived from the Mn2+-mediated activation with respect to absorption spectra and the lack of enoyl reductase activity. The free inhibitors obtained from these two complexes were also identical in their absorption spectra, HPLC retention times, and mass spectra. These results indicate that the same InhA inhibitor was generated by either the slow Mn2+-mediated or the fast KatG-catalyzed INH activation. Both the free and the InhA-bound inhibitor were sufficiently stable to allow the isolation and the subsequent characterization of the inhibitor. The complexes derived from both activation processes were indistinguishable from those reconstituted from InhA and the isolated free inhibitor with respect to absorption spectra. The binding of the inhibitor to InhA was apparently very tight with a dissociation constant estimated to be lower than 0.4 nm. The free inhibitor has absorption peaks at 260 and 326 nm (Fig. 2 A). In comparison with NADH, the 326-nm peak of the inhibitor is stronger in absorptivity (ε326 about 6900m−1 cm−1) and in a shorter wavelength range than the NADH 340-nm peak (ε340 6200m−1 cm−1). Upon binding to InhA, the inhibitor 326-nm peak was blue-shifted, but the complex still showed a pronounced shoulder around 320 nm (Fig. 2 A). The characteristic absorption around 320 nm provides a very useful reporting signal for monitoring the formation and isolation of the inhibitor. The spectral changes resulting from the inhibitor binding by InhA, especially the ΔA 353 (Fig.2 B), also provide a convenient means for investigating the binding of the inhibitor by InhA. Using this method, the KatG S315T mutant frequently encountered in INH-resistant M. tuberculosis isolates has been shown to fail to catalyze the formation of the InhA inhibitor. 2B. Lei and S.-C. Tu, unpublished results. We believe that the methodology developed in this report can also be applied to a recently identified INH target KasA (17.Mdluli K. Slayden R.A. Zhu Y. Ramaswamy S. Pan X. Mead D. Crane D.D. Musser J.M. Barry III, C.E. Science. 1998; 280: 1607-1610Crossref PubMed Scopus (373) Google Scholar) for further verification and investigation of the inactivation mechanism. There are some debates about whether InhA is a primary target of activated INH in M. tuberculosis (29.Mdluli K. Sherman D.R. Hickey M.J. Kreiswirth B.N. Morris S. Stover C.K. Barry III, C.E. J. Infect. Dis. 1996; 174: 1085-1090Crossref PubMed Scopus (102) Google Scholar), in part due to the infrequency of mutation in the inhA gene in INH-resistantM. tuberculosis strains. Particularly, the S92A mutation of InhA, which confers high INH resistance in M. smegmatis(15.Banerjee A. Dubnau E. Quemard A. Balasubramanian V. Um K.S. Wilson T. Collins D. de Lisle G. Jacobs W.R. Science. 1994; 263: 227-230Crossref PubMed Scopus (1207) Google Scholar), has not been encountered in M. tuberculosis isolates from INH-resistant patients. Our findings support InhA as a primary target of INH action in M. tuberculosis. KatG catalyzes the formation of the InhA inhibitor. The extremely high affinity of the inhibitor to InhA (K d < 0.4 nm) is consistent with the high susceptibility of M. tuberculosisto INH. There are two possibilities for the lack of the S92A InhA allele in M. tuberculosis. One is that the selection pressure is not high enough for the mutation. M. smegmatis(which does not cause tuberculosis) and M. tuberculosis are sensitive to INH with minimum inhibitory concentrations of > 5 and 0.01–0.02 μg/ml, respectively. The M. smegmatis InhA S92A mutant confers INH resistance of minimum inhibitory concentrations > 50 μg/ml, whereas most of clinical INH-resistant M. tuberculosis isolates have minimum inhibitory concentrations of 1–5 μg/ml. In clinical treatments, INH was absorbed by the gut to reach peak levels of 3–7 μg/ml in 1–2 h after a usual oral dosage of 300 mg (30.Holdiness M.R. Clin. Pharmacokinet. 1984; 9: 511-544Crossref PubMed Scopus (194) Google Scholar). At such an in vivolevel of INH, the InhA S92A mutation of M. tuberculosiswould not be effectively selected. The other possibility is that, because the InhA enzymes from M. tuberculosis and M. smegmatis have 11.7% nonidentity in their amino acid sequences, the M. tuberculosis InhA S92A mutant may be distinct from the corresponding M. smegmatis mutant in remaining sensitive to the inhibitor. This latter possibility is under current investigation. The M. smegmatis KatG has been shown to require Mn2+ for the activation of INH for the inhibition of InhA (19.Zabinski R.F. Blanchard J.S. J. Am. Chem. Soc. 1997; 119: 2331-2332Crossref Scopus (59) Google Scholar), possibly by converting Mn2+ to Mn3+, which in turn oxidizes INH (31.Magliozzo R.S. Marcinkeviciene J.A. J. Biol. Chem. 1997; 272: 8867-8870Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). We found that, although Mn2+ enhanced the INH activation by M. tuberculosis KatG, the M. tuberculosis KatG can efficiently activate INH without exogenously added Mn2+. We did not find any detectable amount of Mn2+ in the Pi buffer, the purified InhA, or the purified KatG used in the activation reaction. Therefore, Mn2+ is apparently not essential to the activation of INH by M. tuberculosis KatG. The two KatG enzymes from M. tuberculosis and M. smegmatis are thus different in their modes of the INH activation. Such a difference might be related to the differential susceptibilities of M. tuberculosis and M. smegmatis to INH. Previous in vitro experiments of INH activation all included InhA in the reaction. It is not clear whether InhA is required in addition to KatG for INH activation. A similar level of the InhA inhibitor was generated in the KatG reactions with or without InhA. Therefore, the simultaneous presence of InhA and KatG is not required for the inhibitor production. KatG used in this report had a Met-Asn-Ser tripeptide fused to the first residue Met of the wild type KatG. In comparison with the wild-type KatG, the purified modified enzyme exhibited essentially the same A 408/A 280 ratio, kinetic parameters, the ability to activate INH, and blue shift of the Soret band A 408 (32.Wengenack N.L. Todorovic S., Yu, L. Rusnak F. Biochemistry. 1998; 37: 15825-15834Crossref PubMed Scopus (70) Google Scholar) upon INH binding (not shown). The extra peptide apparently does not significantly change the structure or function of KatG. This is in contrast with another fusion enzyme that had a Met-Glu-Phe-Val tetrapeptide fused to the second residue Pro. This latter modified KatG bound only about 0.5 heme/dimer, in comparison with 2 heme/dimer by the wild type and our modified KatG, and thus had a much lower enzyme activity (33.Nagy J.M. Cass A.E.G. Brown K.A. J. Biol. Chem. 1997; 272: 31265-31271Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). A constitutive expression was adopted to slowly accumulate our enzyme while the other KatG fusion was overexpressed with an inducible system. The difference in the expression strategy could lead to the difference in the incorporation of the heme cofactor. We thank Dr. Peter C. Loewen at University of Manitoba for the generous gift of the katG-deficient strain of E. coli UM262. We are grateful to Vance Jason Styve and Dr. James Meen (University of Houston) for performing the inductively coupled plasma analysis, and to Christopher Prater and Dr. Subbarao Kala (Baylor College of Medicine) for conducting the mass spectrometry analysis. The Perkin-Elmer inductively coupled plasma/5500 emission spectrometer used in this work is part of the TcSUH/MRSEC Shared Facilities supported by the State of Texas through the Texas Center for Superconductivity at the University of Houston and by the National Science Foundation under Award Number DMR-9632667.