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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

Proposed mechanismof activation of ISO by EthA and inhibition of the FAS-II HadAB dehydratase.ISO in the enol form is oxidized by EthA to the sulfenic acid, whichreacts with the sulfhydryl on Cys61 of HadAWT or HadAC105A, thereby inactivating the HadAB dehydratase. In the caseof HadAWT, either in the cell itself or during its isolation,the sulfhydryl of Cys105 further displaces the ISO forming, as shownherein, an additional (oxidized) inactive form of the dehydratase.The peptide backbone is shown in part for HadA.
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fig5: Proposed mechanismof activation of ISO by EthA and inhibition of the FAS-II HadAB dehydratase.ISO in the enol form is oxidized by EthA to the sulfenic acid, whichreacts with the sulfhydryl on Cys61 of HadAWT or HadAC105A, thereby inactivating the HadAB dehydratase. In the caseof HadAWT, either in the cell itself or during its isolation,the sulfhydryl of Cys105 further displaces the ISO forming, as shownherein, an additional (oxidized) inactive form of the dehydratase.The peptide backbone is shown in part for HadA.

Mentions: To determine whether the modificationof Cys61 by ISO and TAC affected the activity of HadAB, we next comparedthe hydratase activity of untreated HadAC105AHadB and theHadAC105AHadB-drug adducts partially purified from BCGon a trans-2-dodecenoyl-CoA substrate using the spectrophotometricassay described by Sacco et al.13 Notethat although >50% of the HadAC105A-HadB heterodimersrecovered from the drug-treated cells apparently reacted with thedrugs under our experimental conditions (Figure 3A), the samples also contained some unbound and presumably activeenzyme. Results showed that the HadAC105A-HadB enzymesfrom TAC- and ISO-treated cells exhibited only 24 and 7%, respectively,of the activity of HadAC105A-HadB partially purified fromuntreated cells (Figures 3E and S3). Incubation of the protein samples for 4.5h in a solution containing 10 mM DTT to reverse the covalent modificationof Cys61 by the drugs led to recovery of enzymatic activity, albeitto a significantly greater extent in the case of TAC than ISO (whichrecovered 82 and 24%, respectively, of the activity of the non-drug-treatedcontrol) (Figure 3E), most likely reflectingdifferences in the susceptibility of the HadAC105A-HadB-drugcomplexes to the reducing agent. Finally, because of our finding thatthe presence of the second Cys residue (Cys105) in HadA may causethe HadA-drug adducts to be unstable, ultimately causing the formationof a disulfide bond in the protein (as described in Figure 5 for ISO), we tested whether the oxidized form ofHadAB generated in vitro upon treatment of the purified enzyme withdiamide (see the SI and Figure S4) displayedenzymatic activity. The results showed that HadAB-treated diamideexhibited only 22.5% of the activity of the untreated enzyme.


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)

Proposed mechanismof activation of ISO by EthA and inhibition of the FAS-II HadAB dehydratase.ISO in the enol form is oxidized by EthA to the sulfenic acid, whichreacts with the sulfhydryl on Cys61 of HadAWT or HadAC105A, thereby inactivating the HadAB dehydratase. In the caseof HadAWT, either in the cell itself or during its isolation,the sulfhydryl of Cys105 further displaces the ISO forming, as shownherein, an additional (oxidized) inactive form of the dehydratase.The peptide backbone is shown in part for HadA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4401429&req=5

fig5: Proposed mechanismof activation of ISO by EthA and inhibition of the FAS-II HadAB dehydratase.ISO in the enol form is oxidized by EthA to the sulfenic acid, whichreacts with the sulfhydryl on Cys61 of HadAWT or HadAC105A, thereby inactivating the HadAB dehydratase. In the caseof HadAWT, either in the cell itself or during its isolation,the sulfhydryl of Cys105 further displaces the ISO forming, as shownherein, an additional (oxidized) inactive form of the dehydratase.The peptide backbone is shown in part for HadA.
Mentions: To determine whether the modificationof Cys61 by ISO and TAC affected the activity of HadAB, we next comparedthe hydratase activity of untreated HadAC105AHadB and theHadAC105AHadB-drug adducts partially purified from BCGon a trans-2-dodecenoyl-CoA substrate using the spectrophotometricassay described by Sacco et al.13 Notethat although >50% of the HadAC105A-HadB heterodimersrecovered from the drug-treated cells apparently reacted with thedrugs under our experimental conditions (Figure 3A), the samples also contained some unbound and presumably activeenzyme. Results showed that the HadAC105A-HadB enzymesfrom TAC- and ISO-treated cells exhibited only 24 and 7%, respectively,of the activity of HadAC105A-HadB partially purified fromuntreated cells (Figures 3E and S3). Incubation of the protein samples for 4.5h in a solution containing 10 mM DTT to reverse the covalent modificationof Cys61 by the drugs led to recovery of enzymatic activity, albeitto a significantly greater extent in the case of TAC than ISO (whichrecovered 82 and 24%, respectively, of the activity of the non-drug-treatedcontrol) (Figure 3E), most likely reflectingdifferences in the susceptibility of the HadAC105A-HadB-drugcomplexes to the reducing agent. Finally, because of our finding thatthe presence of the second Cys residue (Cys105) in HadA may causethe HadA-drug adducts to be unstable, ultimately causing the formationof a disulfide bond in the protein (as described in Figure 5 for ISO), we tested whether the oxidized form ofHadAB generated in vitro upon treatment of the purified enzyme withdiamide (see the SI and Figure S4) displayedenzymatic activity. The results showed that HadAB-treated diamideexhibited only 22.5% of the activity of the untreated enzyme.

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