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Covalent modification of the Mycobacterium tuberculosis FAS-II dehydratase by Isoxyl and Thiacetazone.

Grzegorzewicz AE, Eynard N, Quémard A, North EJ, Margolis A, Lindenberger JJ, Jones V, Korduláková J, Brennan PJ, Lee RE, Ronning DR, McNeil MR, Jackson M - ACS Infect Dis (2015)

Bottom Line: We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity.Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs.Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.

ABSTRACT

Isoxyl and Thiacetazone are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation and the nature of their activated forms remained unknown. We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity. Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs. Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

No MeSH data available.


Related in: MedlinePlus

Structuralmodel of M. tuberculosis HadAB modified at HadA-C61.(A) The heterodimeric HadAB complex is shown as a ribbon diagram withHadA in gray and HadB in orange. Carbon atoms and bonds of each proteinmolecule are shown in the respective ribbon color. The carbon atomsof TAC are shown in bronze. All other atoms are colored by CPK. HadB-D36and HadB-H41, essential active site residues, highlight the HadABactive site. The atoms of HadA-C61 are shown as spheres. HadA-C61is at the end of an internal channel that extends from the enzymesurface near active site residues HadB-D36 and HadB-H41. (B) The predictedinteractions supporting complex formation between TAC and HadAB areshown. This model exhibits significant chemical complementary betweenthe predicted lipid-binding pocket of HadAB and the TAC molecule.(C) The predicted interactions supporting complex formation betweenISO and HadAB are shown in the same orientation as in panel B. Inaddition to the complementary interactions observed in the TAC model,the ISO model exhibits significant van der Waals interactions betweenthe atoms of the second phenyl isopentyl ether arm of ISO and theatoms forming a hydrophobic pocket in HadAB abutting HadA-C61.
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fig6: Structuralmodel of M. tuberculosis HadAB modified at HadA-C61.(A) The heterodimeric HadAB complex is shown as a ribbon diagram withHadA in gray and HadB in orange. Carbon atoms and bonds of each proteinmolecule are shown in the respective ribbon color. The carbon atomsof TAC are shown in bronze. All other atoms are colored by CPK. HadB-D36and HadB-H41, essential active site residues, highlight the HadABactive site. The atoms of HadA-C61 are shown as spheres. HadA-C61is at the end of an internal channel that extends from the enzymesurface near active site residues HadB-D36 and HadB-H41. (B) The predictedinteractions supporting complex formation between TAC and HadAB areshown. This model exhibits significant chemical complementary betweenthe predicted lipid-binding pocket of HadAB and the TAC molecule.(C) The predicted interactions supporting complex formation betweenISO and HadAB are shown in the same orientation as in panel B. Inaddition to the complementary interactions observed in the TAC model,the ISO model exhibits significant van der Waals interactions betweenthe atoms of the second phenyl isopentyl ether arm of ISO and theatoms forming a hydrophobic pocket in HadAB abutting HadA-C61.

Mentions: Different mechanisms may account for the inhibition of thedehydratase activity of FAS-II upon covalent binding of ISO and TACto the Cys61 residue of HadA. HadA, HadB, and HadC were shown to associatein two functional heterodimers, HadAB and HadBC, where HadB is thecommon subunit carrying the catalytic site.13 In vitro, HadAB displays a greater affinity for shorter fatty acylchains than HadBC, and it is thought that HadAB acts at the earlystages of the elongation of meromycolates, whereas HadBC dehydrateslonger fatty acyl chains in a mechanism reminiscent of that describedfor the FAS-II β-ketoacyl-ACP synthases KasA and KasB.19 HadA and HadC were proposed to play a role inthe stabilization of the acyl-ACP substrates by keeping open the activesite tunnels in HadAB and HadBC.13 On thebasis of the experiment presented in Figure 3A which shows that comparable amounts of HadB coeluted with HadAC105A-His in the untreated and drug-treated samples, it appearsthat the Cys61 modification of HadA by the drugs does not result inthe dissociation of the HadAB heterodimer. That the drugs covalentlybind to the catalytic subunit HadB or to HadC or cause the dissociationof the HadBC heterodimer is also not supported by our data (Figures 2, 3 and S5). Instead, and in light of the structural modeling of HadABpresented in Figure 6, it is likely that themodification of Cys61 by ISO and TAC blocks the acyl-ACPs’access to the acyl-binding channel located at the interface of HadAand HadB (Figure 6A). Indeed, in addition toaffording disulfide bond formation between the thiol of HadA-C61 andthe tested compounds, this channel is likely to accommodate all ofTAC and one of the two phenyl isopentyl ether arms of ISO (Figure 6B,C). Multiple interactions between amino acid sidechains forming this pocket appear to promote binding of either drugas outlined in Figure 6 and the SI. The resulting inability of the HadAB-ISOor HadAB-TAC adducts to dehydrate early meromycolate precursors wouldinterrupt their elongation by FAS-II and explain the complete shut-downof mycolic acid biosynthesis that accompanies the buildup of early(C8–C22) 3-hydroxy meromycolic acidsobserved in drug-treated cells.8 Clearly,the structural characterization of the FAS-II dehydratases alone andin complex with ISO and TAC will be crucial to the further understandingof their catalytic activity and inhibition by both drugs.


Covalent modification of the Mycobacterium tuberculosis FAS-II dehydratase by Isoxyl and Thiacetazone.

Grzegorzewicz AE, Eynard N, Quémard A, North EJ, Margolis A, Lindenberger JJ, Jones V, Korduláková J, Brennan PJ, Lee RE, Ronning DR, McNeil MR, Jackson M - ACS Infect Dis (2015)

Structuralmodel of M. tuberculosis HadAB modified at HadA-C61.(A) The heterodimeric HadAB complex is shown as a ribbon diagram withHadA in gray and HadB in orange. Carbon atoms and bonds of each proteinmolecule are shown in the respective ribbon color. The carbon atomsof TAC are shown in bronze. All other atoms are colored by CPK. HadB-D36and HadB-H41, essential active site residues, highlight the HadABactive site. The atoms of HadA-C61 are shown as spheres. HadA-C61is at the end of an internal channel that extends from the enzymesurface near active site residues HadB-D36 and HadB-H41. (B) The predictedinteractions supporting complex formation between TAC and HadAB areshown. This model exhibits significant chemical complementary betweenthe predicted lipid-binding pocket of HadAB and the TAC molecule.(C) The predicted interactions supporting complex formation betweenISO and HadAB are shown in the same orientation as in panel B. Inaddition to the complementary interactions observed in the TAC model,the ISO model exhibits significant van der Waals interactions betweenthe atoms of the second phenyl isopentyl ether arm of ISO and theatoms forming a hydrophobic pocket in HadAB abutting HadA-C61.
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Related In: Results  -  Collection

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fig6: Structuralmodel of M. tuberculosis HadAB modified at HadA-C61.(A) The heterodimeric HadAB complex is shown as a ribbon diagram withHadA in gray and HadB in orange. Carbon atoms and bonds of each proteinmolecule are shown in the respective ribbon color. The carbon atomsof TAC are shown in bronze. All other atoms are colored by CPK. HadB-D36and HadB-H41, essential active site residues, highlight the HadABactive site. The atoms of HadA-C61 are shown as spheres. HadA-C61is at the end of an internal channel that extends from the enzymesurface near active site residues HadB-D36 and HadB-H41. (B) The predictedinteractions supporting complex formation between TAC and HadAB areshown. This model exhibits significant chemical complementary betweenthe predicted lipid-binding pocket of HadAB and the TAC molecule.(C) The predicted interactions supporting complex formation betweenISO and HadAB are shown in the same orientation as in panel B. Inaddition to the complementary interactions observed in the TAC model,the ISO model exhibits significant van der Waals interactions betweenthe atoms of the second phenyl isopentyl ether arm of ISO and theatoms forming a hydrophobic pocket in HadAB abutting HadA-C61.
Mentions: Different mechanisms may account for the inhibition of thedehydratase activity of FAS-II upon covalent binding of ISO and TACto the Cys61 residue of HadA. HadA, HadB, and HadC were shown to associatein two functional heterodimers, HadAB and HadBC, where HadB is thecommon subunit carrying the catalytic site.13 In vitro, HadAB displays a greater affinity for shorter fatty acylchains than HadBC, and it is thought that HadAB acts at the earlystages of the elongation of meromycolates, whereas HadBC dehydrateslonger fatty acyl chains in a mechanism reminiscent of that describedfor the FAS-II β-ketoacyl-ACP synthases KasA and KasB.19 HadA and HadC were proposed to play a role inthe stabilization of the acyl-ACP substrates by keeping open the activesite tunnels in HadAB and HadBC.13 On thebasis of the experiment presented in Figure 3A which shows that comparable amounts of HadB coeluted with HadAC105A-His in the untreated and drug-treated samples, it appearsthat the Cys61 modification of HadA by the drugs does not result inthe dissociation of the HadAB heterodimer. That the drugs covalentlybind to the catalytic subunit HadB or to HadC or cause the dissociationof the HadBC heterodimer is also not supported by our data (Figures 2, 3 and S5). Instead, and in light of the structural modeling of HadABpresented in Figure 6, it is likely that themodification of Cys61 by ISO and TAC blocks the acyl-ACPs’access to the acyl-binding channel located at the interface of HadAand HadB (Figure 6A). Indeed, in addition toaffording disulfide bond formation between the thiol of HadA-C61 andthe tested compounds, this channel is likely to accommodate all ofTAC and one of the two phenyl isopentyl ether arms of ISO (Figure 6B,C). Multiple interactions between amino acid sidechains forming this pocket appear to promote binding of either drugas outlined in Figure 6 and the SI. The resulting inability of the HadAB-ISOor HadAB-TAC adducts to dehydrate early meromycolate precursors wouldinterrupt their elongation by FAS-II and explain the complete shut-downof mycolic acid biosynthesis that accompanies the buildup of early(C8–C22) 3-hydroxy meromycolic acidsobserved in drug-treated cells.8 Clearly,the structural characterization of the FAS-II dehydratases alone andin complex with ISO and TAC will be crucial to the further understandingof their catalytic activity and inhibition by both drugs.

Bottom Line: We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity.Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs.Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

View Article: PubMed Central - PubMed

Affiliation: Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523-1682, USA.

ABSTRACT

Isoxyl and Thiacetazone are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation and the nature of their activated forms remained unknown. We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity. Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs. Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.

No MeSH data available.


Related in: MedlinePlus