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Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors.

Dong Y, Qiu X, Shaw N, Xu Y, Sun Y, Li X, Li J, Rao Z - Protein Cell (2015)

Bottom Line: We show that inhibitors bind in this cavity and protrude into the substrate binding channel.Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site.The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

ABSTRACT
Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

No MeSH data available.


Related in: MedlinePlus

Mechanism of inhibition of enzymatic activity ofMtbHadAB complex. (A) Flavonoids bind in the cavity (gray colored surface representation) that traverses the fatty acid binding channel. An example of how binding of butein (cyan sticks) physically obstructs the placement of fatty acid (modeled as green sticks) in the substrate binding channel is shown. (B) All the three flavonoids (sticks) protrude into the substrate binding channel and perturb binding of the substrate in the active site. The cavity and substrate binding channel are shown as gray colored surface representation. (C) The inhibition model of thiacetazone (TAC, modeled as sticks) on MtbHadAB. TAC is shown covalently attached to Cys61 (shown as cyan colored sticks). C61S mutation is known to confer resistance to TAC. The drug protrudes into the fatty acid binding channel (gray colored surface representation) and occludes binding of fatty acid
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Fig6: Mechanism of inhibition of enzymatic activity ofMtbHadAB complex. (A) Flavonoids bind in the cavity (gray colored surface representation) that traverses the fatty acid binding channel. An example of how binding of butein (cyan sticks) physically obstructs the placement of fatty acid (modeled as green sticks) in the substrate binding channel is shown. (B) All the three flavonoids (sticks) protrude into the substrate binding channel and perturb binding of the substrate in the active site. The cavity and substrate binding channel are shown as gray colored surface representation. (C) The inhibition model of thiacetazone (TAC, modeled as sticks) on MtbHadAB. TAC is shown covalently attached to Cys61 (shown as cyan colored sticks). C61S mutation is known to confer resistance to TAC. The drug protrudes into the fatty acid binding channel (gray colored surface representation) and occludes binding of fatty acid

Mentions: The molecular basis for the inhibition of MtbHadAB can be envisioned based on the structures of MtbHadAB bound with flavonoids. Binding of the flavonoid to MtbHadAB can physically occlude binding of the fatty acid into the substrate binding channel (Fig. 6A). All the three flavonoids protrude into the fatty acid binding channel (Fig. 6B). Notably, they are in proximity of the catalytic amino acids. Therefore, the flavonoids can scuttle binding of the fatty acid or prevent it from binding in a catalytically competent orientation. Amongst the flavonoids tested for inhibition of growth of Mycobacterium bovis BCG strain, butein was the most effective with an MIC value of 157 μmol/L (43 μg/mL) (Brown et al., 2007b). This can be explained by examining the structures of the binary complexes of the flavonoids with MtbHadAB. The O1 hydroxyl group of butein forms a hydrogen bond with the putative catalytic water Wat3 that is hydrogen bonded to both the residues of the His-Asp dyad (Figs. 5C and S7). This interaction is not observed in HCC and fisetin. Interestingly, the IC50 value for the inhibition of FAS II system by fisetin was 54 μg/mL, which was almost twice of that exhibited by HCC and butein. Inspection of the structures of flavonoids bound with MtbHadAB reveals that the obstruction of the fatty acid binding channel by fisetin is the least efficient amongst the three flavonoids. While butein and HCC can obstruct and almost completely block the fatty acid binding channel, fisetin protrudes into the channel; but, it only partially covers the fatty acid binding channel (Fig. 6B). Thus, crystal structures of MtbHadAB bound with flavonoids illustrate that butein, fisetin, and HCC exert their inhibitory effect by obstructing the placement of the fatty acid into the substrate binding site of MtbHadAB.Figure 6


Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors.

Dong Y, Qiu X, Shaw N, Xu Y, Sun Y, Li X, Li J, Rao Z - Protein Cell (2015)

Mechanism of inhibition of enzymatic activity ofMtbHadAB complex. (A) Flavonoids bind in the cavity (gray colored surface representation) that traverses the fatty acid binding channel. An example of how binding of butein (cyan sticks) physically obstructs the placement of fatty acid (modeled as green sticks) in the substrate binding channel is shown. (B) All the three flavonoids (sticks) protrude into the substrate binding channel and perturb binding of the substrate in the active site. The cavity and substrate binding channel are shown as gray colored surface representation. (C) The inhibition model of thiacetazone (TAC, modeled as sticks) on MtbHadAB. TAC is shown covalently attached to Cys61 (shown as cyan colored sticks). C61S mutation is known to confer resistance to TAC. The drug protrudes into the fatty acid binding channel (gray colored surface representation) and occludes binding of fatty acid
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491049&req=5

Fig6: Mechanism of inhibition of enzymatic activity ofMtbHadAB complex. (A) Flavonoids bind in the cavity (gray colored surface representation) that traverses the fatty acid binding channel. An example of how binding of butein (cyan sticks) physically obstructs the placement of fatty acid (modeled as green sticks) in the substrate binding channel is shown. (B) All the three flavonoids (sticks) protrude into the substrate binding channel and perturb binding of the substrate in the active site. The cavity and substrate binding channel are shown as gray colored surface representation. (C) The inhibition model of thiacetazone (TAC, modeled as sticks) on MtbHadAB. TAC is shown covalently attached to Cys61 (shown as cyan colored sticks). C61S mutation is known to confer resistance to TAC. The drug protrudes into the fatty acid binding channel (gray colored surface representation) and occludes binding of fatty acid
Mentions: The molecular basis for the inhibition of MtbHadAB can be envisioned based on the structures of MtbHadAB bound with flavonoids. Binding of the flavonoid to MtbHadAB can physically occlude binding of the fatty acid into the substrate binding channel (Fig. 6A). All the three flavonoids protrude into the fatty acid binding channel (Fig. 6B). Notably, they are in proximity of the catalytic amino acids. Therefore, the flavonoids can scuttle binding of the fatty acid or prevent it from binding in a catalytically competent orientation. Amongst the flavonoids tested for inhibition of growth of Mycobacterium bovis BCG strain, butein was the most effective with an MIC value of 157 μmol/L (43 μg/mL) (Brown et al., 2007b). This can be explained by examining the structures of the binary complexes of the flavonoids with MtbHadAB. The O1 hydroxyl group of butein forms a hydrogen bond with the putative catalytic water Wat3 that is hydrogen bonded to both the residues of the His-Asp dyad (Figs. 5C and S7). This interaction is not observed in HCC and fisetin. Interestingly, the IC50 value for the inhibition of FAS II system by fisetin was 54 μg/mL, which was almost twice of that exhibited by HCC and butein. Inspection of the structures of flavonoids bound with MtbHadAB reveals that the obstruction of the fatty acid binding channel by fisetin is the least efficient amongst the three flavonoids. While butein and HCC can obstruct and almost completely block the fatty acid binding channel, fisetin protrudes into the channel; but, it only partially covers the fatty acid binding channel (Fig. 6B). Thus, crystal structures of MtbHadAB bound with flavonoids illustrate that butein, fisetin, and HCC exert their inhibitory effect by obstructing the placement of the fatty acid into the substrate binding site of MtbHadAB.Figure 6

Bottom Line: We show that inhibitors bind in this cavity and protrude into the substrate binding channel.Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site.The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

View Article: PubMed Central - PubMed

Affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.

ABSTRACT
Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2',4,4'-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.

No MeSH data available.


Related in: MedlinePlus