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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

Chemical structures of compounds of the current study related to inhibition ofMtbHadAB. Thiacetazone (TAC) and isoxyl (ISO) have a sulfur containing group that could form a di-sulfide bond with Cys61 of HadA. Mycobacterium tuberculosis strains with C61S mutation in HadA are resistant to TAC and ISO
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Fig1: Chemical structures of compounds of the current study related to inhibition ofMtbHadAB. Thiacetazone (TAC) and isoxyl (ISO) have a sulfur containing group that could form a di-sulfide bond with Cys61 of HadA. Mycobacterium tuberculosis strains with C61S mutation in HadA are resistant to TAC and ISO

Mentions: The β-hydroxyacyl-ACP dehydratase (Had) catalyzes the third step in the fatty acid elongation cycle by dehydrating β-hydroxyacyl-ACP to trans-2-enoyl-ACP (Fig. S1), which is the last piece to be identified in the mycobacterial FAS-II and exsits only in Corynebacterineae (Sacco et al., 2007). HadAB would take part, like KasA, in the early FA elongation cycles, leading to the formation of the intermediate-size (C32–C42) meromycolic chains, while HadBC, like KasB, would elongate further the intermediate-size meromycolic chains to full-size molecules (C52–C64) during the late elongation cycles (Gao et al., 2003; Sacco et al., 2007). Previously, flavonoid inhibitors targeting HadB (Rv0636) were shown to disrupt the biosynthesis of fatty acids, resulting in the depletion of the mycolic acid content of the Mycobacteria. Consequently, these flavonoids were shown to effectively inhibit the growth of Mycobacterium bovis BCG (Brown et al., 2007a). Besides flavonoids, two pro-drugs, isoxyl (ISO) and thiacetazone (TAC) (Fig. 1) used in the clinical treatment of tuberculosis, are also known to exert their anti-mycobacterial effect by stalling the dehydration step of the FAS-II elongation cycle (Belardinelli and Morbidoni, 2012; Coxon et al., 2013; Grzegorzewicz et al., 2012). Both these pro-drugs undergo activation by monooxygenase EthA, for unleashing their anti-mycobacterial potential (Dover et al., 2007; Kordulakova et al., 2007; Nishida and Ortiz de Montellano, 2011). How these drugs disrupt the dehydratase activity of the FAS-II system has remained an enigma for years. A lack of understanding of the molecular basis of this inhibition has been a major bottleneck in the development of next generation of drugs essential for targeting the mycolic acid component of mycobacteria.Figure 1


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)

Chemical structures of compounds of the current study related to inhibition ofMtbHadAB. Thiacetazone (TAC) and isoxyl (ISO) have a sulfur containing group that could form a di-sulfide bond with Cys61 of HadA. Mycobacterium tuberculosis strains with C61S mutation in HadA are resistant to TAC and ISO
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4491049&req=5

Fig1: Chemical structures of compounds of the current study related to inhibition ofMtbHadAB. Thiacetazone (TAC) and isoxyl (ISO) have a sulfur containing group that could form a di-sulfide bond with Cys61 of HadA. Mycobacterium tuberculosis strains with C61S mutation in HadA are resistant to TAC and ISO
Mentions: The β-hydroxyacyl-ACP dehydratase (Had) catalyzes the third step in the fatty acid elongation cycle by dehydrating β-hydroxyacyl-ACP to trans-2-enoyl-ACP (Fig. S1), which is the last piece to be identified in the mycobacterial FAS-II and exsits only in Corynebacterineae (Sacco et al., 2007). HadAB would take part, like KasA, in the early FA elongation cycles, leading to the formation of the intermediate-size (C32–C42) meromycolic chains, while HadBC, like KasB, would elongate further the intermediate-size meromycolic chains to full-size molecules (C52–C64) during the late elongation cycles (Gao et al., 2003; Sacco et al., 2007). Previously, flavonoid inhibitors targeting HadB (Rv0636) were shown to disrupt the biosynthesis of fatty acids, resulting in the depletion of the mycolic acid content of the Mycobacteria. Consequently, these flavonoids were shown to effectively inhibit the growth of Mycobacterium bovis BCG (Brown et al., 2007a). Besides flavonoids, two pro-drugs, isoxyl (ISO) and thiacetazone (TAC) (Fig. 1) used in the clinical treatment of tuberculosis, are also known to exert their anti-mycobacterial effect by stalling the dehydration step of the FAS-II elongation cycle (Belardinelli and Morbidoni, 2012; Coxon et al., 2013; Grzegorzewicz et al., 2012). Both these pro-drugs undergo activation by monooxygenase EthA, for unleashing their anti-mycobacterial potential (Dover et al., 2007; Kordulakova et al., 2007; Nishida and Ortiz de Montellano, 2011). How these drugs disrupt the dehydratase activity of the FAS-II system has remained an enigma for years. A lack of understanding of the molecular basis of this inhibition has been a major bottleneck in the development of next generation of drugs essential for targeting the mycolic acid component of mycobacteria.Figure 1

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