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

Yu Dong, Xiaodi Qiu, Neil Shaw, Yueyang Xu, Yuna Sun, Xuemei Li, Jun Li, Zihe Rao

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Protein Cell ›› 2015, Vol. 6 ›› Issue (7) : 504-517. DOI: 10.1007/s13238-015-0181-1
RESEARCH ARTICLE

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

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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.

Keywords

Mycobacterium tuberculosis / hotdog fold / mycolic acid / dehydratase / flavonoid / thiacetazone / isoxyl

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Yu Dong, Xiaodi Qiu, Neil Shaw, Yueyang Xu, Yuna Sun, Xuemei Li, Jun Li, Zihe Rao. Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors. Protein Cell, 2015, 6(7): 504‒517 https://doi.org/10.1007/s13238-015-0181-1

References

[1]
Asselineau C, Asselineau J, Lanéelle G, Lanéelle MA (2002) The biosynthesis of mycolic acids by Mycobacteria: current and alternative hypotheses. Prog Lipid Res41: 501-523
CrossRef Google scholar
[2]
Banerjee A, Dubnau E, Quemard A, Balasubramanian V, Um KS, Wilson T, Collins D, de Lisle G, Jacobs WR Jr (1994) inhA, a gene encoding a target for isoniazid and ethionamide in Mycobacterium tuberculosis. Science263: 227-230
CrossRef Google scholar
[3]
Belardinelli JM, Morbidoni HR (2012) Mutations in the essential FAS II beta-hydroxyacyl ACP dehydratase complex confer resistance to thiacetazone in Mycobacterium tuberculosis and Mycobacterium kansasii. Mol Microbiol86: 568-579
CrossRef Google scholar
[4]
Benning MM, Wesenberg G, Liu R, Taylor KL, Dunaway-Mariano D, Holden HM (1998) The three-dimensional structure of 4-hydroxybenzoyl-CoA thioesterase from Pseudomonas sp. Strain CBS-3. J Biol Chem273: 33572-33579
CrossRef Google scholar
[5]
Bhatt A, Molle V, Besra GS, Jacobs WR Jr, Kremer L (2007) The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development. Mol Microbiol64: 1442-1454
CrossRef Google scholar
[6]
Bloch K (1977) Control mechanisms for fatty acid synthesis in Mycobacterium smegmatis. Adv Enzymol Relat Areas Mol Biol45: 1-84
CrossRef Google scholar
[7]
Brown AK, Bhatt A, Singh A, Saparia E, Evans AF, Besra GS (2007a) Identification of the dehydratase component of the mycobacterial mycolic acid-synthesizing fatty acid synthase-II complex. Microbiology153: 4166-4173
CrossRef Google scholar
[8]
Brown AK, Papaemmanouil A, Bhowruth V, Bhatt A, Dover LG, Besra GS (2007b) Flavonoid inhibitors as novel antimycobacterial agents targeting Rv0636, a putative dehydratase enzyme involved in Mycobacterium tuberculosis fatty acid synthase II. Microbiology153: 3314-3322
CrossRef Google scholar
[9]
Campbell JW, Cronan JE Jr (2001) Bacterial fatty acid biosynthesis: targets for antibacterial drug discovery. Annu Rev Microbiol55: 305-332
CrossRef Google scholar
[10]
Cantaloube S, Veyron-Churlet R, Haddache N, Daffé M, Zerbib D (2011) The Mycobacterium tuberculosis FAS-II dehydratases and methyltransferases define the specificity of the mycolic acid elongation complexes. PLoS One6: e29564
CrossRef Google scholar
[11]
Carel C, Nukdee K, Cantaloube S, Bonne M, Diagne CT, Laval F, Zerbib D (2014) Mycobacterium tuberculosis proteins involved in mycolic acid synthesis and transport localize dynamically to the old growing pole and septum. PLoS One9: e97148
CrossRef Google scholar
[12]
Castell A, Johansson P, Unge T, Jones TA, Backbro K (2005) Rv0216, a conserved hypothetical protein from Mycobacterium tuberculosis that is essential for bacterial survival during infection, has a double hotdog fold. Protein Sci14: 1850-1862
CrossRef Google scholar
[13]
Comas I, Gagneux S (2009) The past and future of tuberculosis research. PLoS Pathog5: e1000600
CrossRef Google scholar
[14]
Coxon GD, Craig D, Corrales RM, Vialla E, Gannoun-Zaki L, Kremer L (2013) Synthesis, antitubercular activity and mechanism of resistance of highly effective thiacetazone analogues. PLoS One8: e53162
CrossRef Google scholar
[15]
Dillon SC, Bateman A(2004) TheHotdog fold: wrapping upa superfamily of thioesterases and dehydratases. BMC Bioinform5: 109
CrossRef Google scholar
[16]
Dover LG, Alahari A, Gratraud P, Gomes JM, Bhowruth V, Reynolds RC, Besra GS, Kremer L (2007) EthA, a common activator of thiocarbamide-containing drugs acting on different mycobacterial targets. Antimicrob Agents Chemother51: 1055-1063
CrossRef Google scholar
[17]
Echols N, Grosse-Kunstleve RW, Afonine PV, Bunkoczi G, Chen VB, Headd JJ, McCoy AJ, Moriarty NW, Read RJ, Richardson DC (2012) Graphical tools for macromolecular crystallography in PHENIX. J Appl Crystallogr45: 581-586
CrossRef Google scholar
[18]
Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr60: 2126-2132
CrossRef Google scholar
[19]
Gannoun-Zaki L, Alibaud L, Kremer L (2013) Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis. Antimicrob Agents Chemother57: 629-632
CrossRef Google scholar
[20]
Gao LY, Laval F, Lawson EH, Groger RK, Woodruff A, Morisaki JH, Cox JS, Daffe M, Brown EJ (2003) Requirement for kasB in Mycobacterium mycolic acid biosynthesis, cell wall impermeability and intracellular survival: implications for therapy. Mol Microbiol49: 1547-1563
CrossRef Google scholar
[21]
Goldberg DE, Siliciano RF, Jacobs WR Jr (2012) Outwitting evolution: fighting drug-resistant TB, malaria, and HIV. Cell148: 1271-1283
CrossRef Google scholar
[22]
Grzegorzewicz AE, Kordulakova J, Jones V, Born SE, Belardinelli JM, Vaquie A, Gundi VA, Madacki J, Slama N, Laval F (2012) A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone. J Biol Chem287: 38434-38441
CrossRef Google scholar
[23]
He L, Zhang L, Liu X, Li X, Zheng M, Li H, Yu K, Chen K, Shen X, Jiang H (2009) Discovering potent inhibitors against the beta-hydroxyacyl-acyl carrier protein dehydratase (FabZ) of Helicobacter pylori: structure-based design, synthesis, bioassay, and crystal structure determination. J Med Chem52: 2465-2481
CrossRef Google scholar
[24]
Holm L, Rosenstrom P (2010) Dali server: conservation mapping in 3D. Nucleic Acids Res38: W545-W549
CrossRef Google scholar
[25]
Jackson M, McNeil MR, Brennan PJ (2013) Progress in targeting cell envelope biogenesis in Mycobacterium tuberculosis. Future Microbiol8: 855-875
CrossRef Google scholar
[26]
Jenni S, Leibundgut M, Boehringer D, Frick C, Mikolasek B, Ban N (2007) Structure of fungal fatty acid synthase and implications for iterative substrate shuttling. Science316: 254-261
CrossRef Google scholar
[27]
Jiang D, Zhang Q, Zheng Q, Zhou H, Jin J, Zhou W, Bartlam M, Rao Z (2014) Structural analysis of Mycobacterium tuberculosis ATPbinding cassette transporter subunit UgpB reveals specificity for glycerophosphocholine. FEBS J281: 331-341
CrossRef Google scholar
[28]
Kimber MS, Martin F, Lu Y, Houston S, Vedadi M, Dharamsi A, Fiebig KM, Schmid M, Rock CO (2004) The structure of (3R)-hydroxyacyl-acyl carrier protein dehydratase (FabZ) from Pseudomonas aeruginosa. J Biol Chem279: 52593-52602
CrossRef Google scholar
[29]
Kirkpatrick AS, Yokoyama T, Choi KJ, Yeo HJ (2009) Campylobacter jejuni fatty acid synthase II: structural and functional analysis of beta-hydroxyacyl-ACP dehydratase (FabZ). Biochem Biophys Res Commun380: 407-412
CrossRef Google scholar
[30]
Kordulakova J, Janin YL, Liav A, Barilone N, Dos Vultos T, Rauzier J, Brennan PJ, Gicquel B, Jackson M (2007) Isoxyl activation is required for bacteriostatic activity against Mycobacterium tuberculosis. Antimicrob Agents Chemother51: 3824-3829
CrossRef Google scholar
[31]
Koski MK, Haapalainen AM, Hiltunen JK, Glumoff T (2004) A twodomain structure of one subunit explains unique features of eukaryotic hydratase 2. J Biol Chem279: 24666-24672
CrossRef Google scholar
[32]
Koski KM, Haapalainen AM, Hiltunen JK, Glumoff T (2005) Crystal structure of 2-enoyl-CoA hydratase 2 from human peroxisomal multifunctional enzyme type 2. J Mol Biol345: 1157-1169
CrossRef Google scholar
[33]
Kostrewa D, Winkler FK, Folkers G, Scapozza L, Perozzo R (2005) The crystal structure of PfFabZ, the unique beta-hydroxyacyl-ACP dehydratase involved in fatty acid biosynthesis of Plasmodium falciparum. Protein Sci14: 1570-1580
CrossRef Google scholar
[34]
Kremer L, Douglas JD, Baulard AR, Morehouse C, Guy MR, Alland D, Dover LG, Lakey JH, Jacobs WR Jr, Brennan PJ (2000) Thiolactomycin and related analogues as novel anti-mycobacterial agents targeting KasA and KasB condensing enzymes in Mycobacterium tuberculosis. J Biol Chem275: 16857-16864
CrossRef Google scholar
[35]
Krissinel E, Henrick K (2007) Inference of macromolecular assemblies from crystalline state. J Mol Biol372: 774-797
CrossRef Google scholar
[36]
Labonte JW, Townsend CA (2013) Active site comparisons and catalytic mechanisms of the hot dog superfamily. Chem Rev113: 2182-2204
CrossRef Google scholar
[37]
Leesong M, Henderson BS, Gillig JR, Schwab JM, Smith JL (1996) Structure of a dehydratase-isomerase from the bacterial pathway for biosynthesis of unsaturated fatty acids: two catalytic activities in one active site. Structure4: 253-264
CrossRef Google scholar
[38]
Leibundgut M, Jenni S, Frick C, Ban N (2007) Structural basis for substrate delivery by acyl carrier protein in the yeast fatty acid synthase. Science316: 288-290
CrossRef Google scholar
[39]
Marrakchi H, Laneelle MA, Daffe M (2014) Mycolic acids: structures, biosynthesis, and beyond. Chem Biol21: 67-85
CrossRef Google scholar
[40]
Matthews BW (1968) Solvent content of protein crystals. J Mol Biol33: 491-497
CrossRef Google scholar
[41]
McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. J Appl Crystallogr40: 658-674
CrossRef Google scholar
[42]
Mdluli K, Slayden RA, Zhu Y, Ramaswamy S, Pan X, Mead D, Crane DD, Musser JM, Barry CE 3rd (1998) Inhibition of a Mycobacterium tuberculosis beta-ketoacyl ACP synthase by isoniazid. Science280: 1607-1610
CrossRef Google scholar
[43]
Moynie L, Leckie SM, McMahon SA, Duthie FG, Koehnke A, Taylor JW, Alphey MS, Brenk R, Smith AD, Naismith JH (2013) Structural insights into the mechanism and inhibition of the beta-hydroxydecanoyl-acyl carrier protein dehydratase from Pseudomonas aeruginosa. J Mol Biol425: 365-377
CrossRef Google scholar
[44]
Nguyen C, Haushalter RW, Lee DJ, Markwick PR, Bruegger J, Caldara-Festin G, Finzel K, Jackson DR, Ishikawa F, O’Dowd B (2014) Trapping the dynamic acyl carrier protein in fatty acid biosynthesis. Nature505: 427-431
CrossRef Google scholar
[45]
Nishida CR, Ortiz de Montellano PR (2011) Bioactivation of antituberculosis thioamide and thiourea prodrugs by bacterial and mammalian flavin monooxygenases. Chem Biol Interact192: 21-25
CrossRef Google scholar
[46]
Otwinowski Z, Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. Academic Press, New York, pp 307-326
CrossRef Google scholar
[47]
Potterton E, Briggs P, Turkenburg M, Dodson E (2003) A graphical user interface to the CCP4 program suite. Acta Crystallogr D Biol Crystallogr59: 1131-1137
CrossRef Google scholar
[48]
Qian L, Ortiz de Montellano PR (2006) Oxidative activation of thiacetazone by the Mycobacterium tuberculosis flavin monooxygenase EtaA and human FMO1 and FMO3. Chem Res Toxicol19: 443-449
CrossRef Google scholar
[49]
Robert X, Gouet P (2014) Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res42: W320-W324
CrossRef Google scholar
[50]
Sacco E, Covarrubias AS, O’Hare HM, Carroll P, Eynard N, Jones TA, Parish T, Daffe M, Backbro K, Quemard A (2007) The missing piece of the type II fatty acid synthase system from Mycobacterium tuberculosis. Proc Natl Acad Sci USA104: 14628-14633
CrossRef Google scholar
[51]
Schneider TR, Sheldrick GM (2002) Substructure solution with SHELXD. Acta Crystallogr D Biol Crystallogr58: 1772-1779
CrossRef Google scholar
[52]
Schrodinger, LLC (2010). The PyMOL Molecular Graphics System, Version 1.3r1.
[53]
Sheldrick GM (2010) Experimental phasing with SHELXC/D/E: combining chain tracing with density modification. Acta Crystallogr D Biol Crystallogr66: 479-485
CrossRef Google scholar
[54]
Swarnamukhi PL, Sharma SK, Bajaj P, Surolia N, Surolia A, Suguna K (2006) Crystal structure of dimeric FabZ of Plasmodium falciparum reveals conformational switching to active hexamers by peptide flips. FEBS Lett580: 2653-2660
CrossRef Google scholar
[55]
Takayama K, Wang C, Besra GS (2005) Pathway to synthesis and processing of mycolic acids in Mycobacterium tuberculosis. Clin Microbiol Rev18: 81-101
CrossRef Google scholar
[56]
Tasdemir D, Lack G, Brun R, Ruedi P, Scapozza L, Perozzo R (2006) Inhibition of Plasmodium falciparum fatty acid biosynthesis: evaluation of FabG, FabZ, and FabI as drug targets for flavonoids. J Med Chem49: 3345-3353
CrossRef Google scholar
[57]
Trivedi OA, Arora P, Sridharan V, Tickoo R, Mohanty D, Gokhale RS (2004) Enzymic activation and transfer of fatty acids as acyladenylates in mycobacteria. Nature428: 441-445
CrossRef Google scholar
[58]
Wang L, Li J, Wang X, Liu W, Zhang XC, Li X, Rao Z (2013) Structure analysis of the extracellular domain reveals disulfide bond forming-protein properties of Mycobacterium tuberculosis Rv2969c. Protein Cell4: 628-640
CrossRef Google scholar
[59]
WHO (2014) Global tuberculosis report 2014 (Geneva, World Health Organization)
[60]
Wong HC, Liu G, Zhang YM, Rock CO, Zheng J (2002) The solution structure of acyl carrier protein from Mycobacterium tuberculosis. J Biol Chem277: 15874-15880
CrossRef Google scholar
[61]
Zhang L, Kong Y, Wu D, Zhang H, Wu J, Chen J, Ding J, Hu L, Jiang H, Shen X (2008a) Three flavonoids targeting the beta-hydroxyacyl-acyl carrier protein dehydratase from Helicobacter pylori: crystal structure characterization with enzymatic inhibition assay. Protein Sci17: 1971-1978
CrossRef Google scholar
[62]
Zhang L, Liu W, Hu T, Du L, Luo C, Chen K, Shen X, Jiang H (2008b) Structural basis for catalytic and inhibitory mechanisms of betahydroxyacyl-acyl carrier protein dehydratase (FabZ). J Biol Chem283: 5370-5379
CrossRef Google scholar
[63]
Zumla A, George A, Sharma V, Herbert N, Masham Baroness, Ilton BM (2013) WHO’s 2013 global report on tuberculosis: successes, threats, and opportunities. Lancet382: 1765-1767
CrossRef Google scholar

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