Biochemistry (Moscow)

, Volume 80, Issue 3, pp 296–309 | Cite as

FoF1-ATP synthase of Streptomyces fradiae ATCC 19609: Structural, biochemical, and functional characterization

  • M. G. Alekseeva
  • T. A. Mironcheva
  • D. A. Mavletova
  • S. M. Elizarov
  • N. V. Zakharevich
  • V. N. DanilenkoEmail author


The patterns of protein phosphorylation in inverted membrane vesicles from the strain Streptomyces fradiae ATCC 19609 were investigated to elucidate the mechanisms of regulation of bacterial membrane bound FoF1-ATP synthase. We found for the first time by two-dimensional gel electrophoresis and mass spectrometry that the β- and b-subunits of the FoF1-ATP synthase complex undergo phosphorylation; 20 proteins with known functions were identified. All eight subunits of FoF1-ATP synthase, i.e. α, β, γ, δ, ɛ, a, b, and c, were cloned into Escherichia coli and expressed as recombinant proteins. Using a crude preparation of serine/threonine protein kinases, we demonstrated the phosphorylation of recombinant γ-, β-, α- and ɛ-subunits. The β-subunit was phosphorylated both as a recombinant protein and in vesicles. Differential phosphorylation of membrane-bound and recombinant proteins can be attributed to different pools of protein kinases in each preparation; in addition, certain steps of FoF1-ATP synthase assembly and function might be accompanied by individual phosphorylation patterns. The structure of the operon containing all subunits and regulatory protein I was identified. The phylogenetic similarity of FoF1-ATP synthase from Streptomyces fradiae ATCC 19609 with the respective proteins in saprophytic and pathogenic (including Mycobacterium tuberculosis) bacteria was investigated. Thus, bacterial serine/threonine protein kinases are important for the regulation of FoF1-ATP synthase. From the practical standpoint, our results provide a basis for designing targeted antibacterial drugs.

Key words

FoF1-ATP synthase Ser/Thr protein kinase inverted membrane vesicles Streptomycetes 



amino acid residue


bis-indolylmaleimide I


base pairs




inhibitor of Ca2+-dependent protein kinases


phenylmethylsulfonyl fluoride


serine/threonine protein kinases


trichloroacetic acid


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Capaldi, R. A., and Aggeler, R. (2002) Mechanism of the F(1)F(0)-type ATP synthase, a biological rotary motor, Trends Biochem. Sci., 27, 154–160.CrossRefPubMedGoogle Scholar
  2. 2.
    Senior, A. E. (2007) ATP synthase: motoring to the finish line, Cell, 130, 220–201.CrossRefPubMedGoogle Scholar
  3. 3.
    Watanabe, R., and Noji, H. (2013) Chemomechanical coupling mechanism of F(1)-ATPase: catalysis and torque generation, FEBS Lett., 587, 1030–1035.CrossRefPubMedGoogle Scholar
  4. 4.
    Okuno, D., Iino, R., and Noji, H. (2011) Rotation and structure of FoF1-ATP synthase, J. Biochem., 149, 655–664.CrossRefPubMedGoogle Scholar
  5. 5.
    Walker, J. E. (2013) The ATP synthase: the understood, the uncertain and the unknown, Biochem. Soc. Trans., 41, 1–16.CrossRefPubMedGoogle Scholar
  6. 6.
    Chi, S. L., and Pizzo, S. V. (2006) Cell surface F1Fo ATP synthase: a new paradigm, Ann. Med., 38, 429–438.CrossRefPubMedGoogle Scholar
  7. 7.
    Skulachev, V. P. (1999) Bacterial energetics at high pH: what happens to the H+ cycle when the extracellular H+ concentration decreases, Novartis Found. Symp., 221, 200–213.PubMedGoogle Scholar
  8. 8.
    Ahmad, Z., Okafor, F., Azim, S., and Laughlin, T. F. (2013) ATP synthase: a molecular therapeutic drug target for antimicrobial and antitumor peptides, Curr. Med. Chem., 20, 1956–1973.CrossRefPubMedGoogle Scholar
  9. 9.
    Lu, P., Lill, H., and Bald, D. (2014) ATP synthase in mycobacteria: special features and implications for a function as drug target, Biochim. Biophys. Acta, 1837, 1208–1218.CrossRefPubMedGoogle Scholar
  10. 10.
    Gras, J. (2013) Bedaquiline for the treatment of pulmonary, multidrug-resistant tuberculosis in adults, Drugs Today (Barcelona), 49, 353–361.Google Scholar
  11. 11.
    Biukovic, G., Basak, S., Manimekalai, M. S., Rishikesan, S., Roessle, M., Dick, T., Rao, S. P., Hunke, C., and Gruber, G. (2013) Variations of subunit {varepsilon} of the Mycobacterium tuberculosis F1Fo ATP synthase and a novel model for mechanism of action of the tuberculosis drug TMC207, Antimicrob. Agents Chemother., 57, 168–176.CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Hensel, M., Deckers-Hebestreit, G., and Altendorf, K. (1991) Purification and characterization of the F1 portion of the ATP synthase (F1Fo) of Streptomyces lividans, Eur. J. Biochem., 202, 1313–1319.CrossRefPubMedGoogle Scholar
  13. 13.
    Hensel, M., Ahmus, H., and Deckers-Hebestreit, G. (1991) The ATP synthase of Streptomyces lividans: characterization and purification of the F1F0 complex, Biochim. Biophys. Acta, 1274, 101–108.CrossRefGoogle Scholar
  14. 14.
    Pagliarani, A., Nesci, S., and Ventrella, V. (2013) Modifiers of the oligomycin sensitivity of the mitochondrial F1F0-ATPase, Mitochondrion, 13, 312–319.CrossRefPubMedGoogle Scholar
  15. 15.
    Shchepina, L. A., Pletjushkina, O. Y., Avetisyan, A. V., Bakeeva, L. E., Fetisova, E. K., Izyumov, D. S., Saprunova, V. B., Vyssokikh, M. Y., Chernyak, B. V., and Skulachev, V. P. (2002) Oligomycin, inhibitor of the F0 part of H+-ATP-synthase, suppresses the TNF-induced apoptosis, Oncogene, 21, 8149–8157.CrossRefPubMedGoogle Scholar
  16. 16.
    Symersky, J., Osowski, D., Walters, D. E., and Mueller, D. M. (2012) Oligomycin frames a common drug-binding site in the ATP synthase, Proc. Natl. Acad. Sci. USA, 109, 13961–13965.CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Alekseeva, M. G., Elizarov, S. M., Bekker, O. B., Lyubimova, I. K., and Danilenko, V. N. (2009) F0F1-ATP synthase of streptomycetes: modulation of activity and sensitivity to oligomycin by serine/threonine protein kinases, Biol. Membr. (Moscow), 26, 41–49.Google Scholar
  18. 18.
    Symersky, J., Pagadala, V., Osowski, D., Krah, A., Meier, T., Faraldo-Gomez, J. D., and Mueller, D. M. (2012) Structure of the c(10) ring of the yeast mitochondrial ATP synthase in the open conformation, Nature Struct. Mol. Biol., 19, 485–491, S1.CrossRefGoogle Scholar
  19. 19.
    Hong, S., and Pedersen, P. L. (2008) ATP synthase and the actions of inhibitors utilized to study its roles in human health, disease, and other scientific areas, Microbiol. Mol. Biol. Rev., 72, 590–641.CrossRefPubMedCentralPubMedGoogle Scholar
  20. 20.
    Lysenkova, L. N., Turchin, K. F., Korolev, A. M., Dezhenkova, L. G., Bekker, O. B., Shtil, A. A., Danilenko, V. N., and Preobrazhenskaya, M. N. (2013) Synthesis and cytotoxicity of oligomycin A derivatives modified in the side chain, Bioorg. Med. Chem., 21, 2918–2924.CrossRefPubMedGoogle Scholar
  21. 21.
    Lysenkova, L. N., Turchin, K. F., Korolev, A. M., Danilenko, V. N., Bekker, O. B., Dezhenkova, L. G., Shtil, A. A., and Preobrazhenskaya, M. N. (2014) Study on retroaldol degradation products of antibiotic oligomycin A, J. Antibiot. (Tokyo), 67, 153–158.CrossRefGoogle Scholar
  22. 22.
    Ko, Y. H., Pan, W., Inoue, C., and Pedersen, P. L. (2002) Signal transduction to mitochondrial ATP synthase: evidence that PDGF-dependent phosphorylation of the deltasubunit occurs in several cell lines, involves tyrosine, and is modulated by lysophosphatidic acid, Mitochondrion, 1, 33–48.CrossRefGoogle Scholar
  23. 23.
    Azarashvily, T. S., Tyynela, J., Baumann, M., Evtodienko, Y. V., and Saris, N. E. (2000) Ca2+-modulated phosphorylation of a low-molecular-mass polypeptide in rat liver mitochondria: evidence that it is identical with subunit c of F0F1-ATPase, Biochem. Biophys. Res. Commun., 270, 741–744.CrossRefPubMedGoogle Scholar
  24. 24.
    Hojlund, K., Wrzesinsk, K., Larsen, P. M., Fey, S. J., Roepstorff, P., Handberg, A., Dela, F., Vinten, J., McCormack, J. G., Reynet, C., and Beck-Nielsen, H. (2003) Proteome analysis reveals phosphorylation of ATP synthase beta-subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes, J. Biol. Chem., 278, 10436–10442.CrossRefPubMedGoogle Scholar
  25. 25.
    Kanekatsu, M., Saito, H., Motohashi, K., and Hisabori, T. (1998) The beta subunit of chloroplast ATP synthase (CF0CF1-ATPase) is phosphorylated by casein kinase II, Biochem. Mol. Biol. Int., 46, 99–105.PubMedGoogle Scholar
  26. 26.
    Prisic, S., Dankwa, S., Schwartz, D., Chou, M. F., Locasale, J. W., Kang, C. M., Bemis, G., Church, G. M., Steen, H., and Husson, R. N. (2010) Extensive phosphorylation with overlapping specificity by Mycobacterium tuberculosis serine/threonine protein kinases, Proc. Natl. Acad. Sci. USA, 107, 7521–7526.CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Inoue, H., Nojima, H., and Okayama, H. (1990) High efficiency transformation of Escherichia coli with plasmids, Gene, 96, 23–28.CrossRefPubMedGoogle Scholar
  28. 28.
    Mierendorf, R., Yeager, K., and Novy, R. (1994) Innovations, Newsletter Novagen, 1, 1–3.Google Scholar
  29. 29.
    Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F., and Hopwood, D. A. (2000) Practical Streptomyces Genetics, The John Innes Foundation, Norwich, United Kingdom.Google Scholar
  30. 30.
    Sambrook, J., Fritsch, E. E., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press.Google Scholar
  31. 31.
    O’Farrell, P. H. (1975) High resolution two-dimensional electrophoresis of proteins, J. Biol. Chem., 250, 4007–4021.PubMedCentralPubMedGoogle Scholar
  32. 32.
    Elizarov, S. M., and Danilenko, V. N. (2001) Multiple phosphorylation of membrane-associated calcium-dependent protein serine/threonine kinase in Streptomyces fradiae, FEMS Microbiol Lett., 202, 135–138.CrossRefPubMedGoogle Scholar
  33. 33.
    Elizarov, S. M., Mironov, V. A., and Danilenko, V. N. (2000) Calcium-induced alterations in the functioning of protein serine/threonine and tyrosine kinases in Streptomyces fradiae cells, IUBMB Life, 50, 139–143.CrossRefPubMedGoogle Scholar
  34. 34.
    Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Res., 22, 4673–4680.CrossRefPubMedCentralPubMedGoogle Scholar
  35. 35.
    Notredame, C., Higgins, D. G., and Heringa, J. (2000) T-Coffee: a novel method for fast and accurate multiple sequence alignment, J. Mol. Biol., 302, 205–217.CrossRefPubMedGoogle Scholar
  36. 36.
    Nicholas, K. B., Nicholas, H. B., Jr., and Deerfield, D. W. (1997) II. GeneDoc: analysis and visualization of genetic variation, EMB News, 4, 14.Google Scholar
  37. 37.
    Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) Basic local alignment search tool, J. Mol. Biol., 215, 403–410.CrossRefPubMedGoogle Scholar
  38. 38.
    Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods, Mol. Biol. Evol., 28, 2731–2739.CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Saitou, N., and Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees, Mol. Biol. Evol., 4, 406–425.PubMedGoogle Scholar
  40. 40.
    Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap, Evolution, 39, 783–791.CrossRefGoogle Scholar
  41. 41.
    Cousin, C., Derouiche, A., Shi, L., Pagot, Y., Poncet, S., and Mijakovic, I. (2013) Protein-serine/threonine/tyrosine kinases in bacterial signaling and regulation, FEMS Microbiol. Lett., 346, 11–19.CrossRefPubMedGoogle Scholar
  42. 42.
    Pereira, S. F., Goss, L., and Dworkin, J. (2011) Eukaryotelike serine/threonine kinases and phosphatases in bacteria, Microbiol. Mol. Biol. Rev., 75, 192–212.CrossRefPubMedCentralPubMedGoogle Scholar
  43. 43.
    Lu, P., Villellas, C., Koul, A., Andries, K., Lill, H., and Bald, D. (2014) The ATP synthase inhibitor bedaquiline interferes with small-molecule efflux in Mycobacterium smegmatis, J. Antibiot. (Tokyo), DOI: 10.1038/ja. 2014.74.Google Scholar
  44. 44.
    Bald, D., and Koul, A. (2013) Advances and strategies in discovery of new antibacterials for combating metabolically resting bacteria, Drug Discov. Today, 18, 250–255.CrossRefPubMedGoogle Scholar
  45. 45.
    Plotnikov, E. Y., Morosanova, M. A., Pevzner, I. B., Zorova, L. D., Manskikh, V. N., Pulkova, N. V., Galkina, S. I., Skulachev, V. P., and Zorov, D. B. (2013) Protective effect of mitochondria-targeted antioxidants in an acute bacterial infection, Proc. Natl. Acad. Sci. USA, 110, 3100–3108.CrossRefGoogle Scholar
  46. 46.
    Skulachev, V. P., Anisimov, V. N., Antonenko, Y. N., Bakeeva, L. E., Chernyak, B. V., Erichev, V. P., Filenko, O. F., Kalinina, N. I., Kapelko, V. I., Kolosova, N. G., Kopnin, B. P., Korshunova, G. A., Lichinitser, M. R., Obukhova, L. A., Pasyukova, E. G., Pisarenko, O. I., Roginsky, V. A., Ruuge, E. K., Senin, I. I., Severina, I. I., Skulachev, M. V., Spivak, I. M., Tashlitsky, V. N., Tkachuk, V. A., Vyssokikh, M. Y., Yaguzhinsky, L. S., and Zorov, D. B. (2009) An attempt to prevent senescence: a mitochondrial approach, Biochim. Biophys. Acta, 1787, 437–461.CrossRefPubMedGoogle Scholar
  47. 47.
    Skulachev, V. P. (2002) Programmed death phenomena: from organelle to organism, Ann. NY Acad. Sci., 959, 214–237.CrossRefPubMedGoogle Scholar
  48. 48.
    Bekker, O. B., Mavletova, D. A., Lyubimova, I. K., Mironcheva, T. A., Shtil, A. A., and Danilenko, V. N. (2012) Induction of programmed lysis of the Streptomyces lividans culture by the inhibitors of eukaryotic-type serine/threonine protein kinases, Mikrobiologiya, 81, 177–184.Google Scholar
  49. 49.
    Bekker, O. B., Alekseeva, M. G., Osolodkin, D. I., Palyulin, V. A., Elizarov, S. M., Zefirov, N. S., and Danilenko, V. N. (2010) Novel test system for the screening of serine-threonine protein kinase inhibitors: E. coli APHVIII/Pk25 construct, Acta Naturae, 2, 126–139.Google Scholar
  50. 50.
    Elizarov, S. M., Alekseeva, M. G., Novikov, F. N., Chilov, G. G., Maslov, D. A., Shtil, A. A., and Danilenko, V. N. (2012) Identification of the sites of phosphorylation of aminoglycoside phosphotransferase VIII from Streptomyces rimosus, Biochemistry (Moscow), 77, 1258–1265.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2015

Authors and Affiliations

  • M. G. Alekseeva
    • 1
  • T. A. Mironcheva
    • 1
  • D. A. Mavletova
    • 1
  • S. M. Elizarov
    • 2
  • N. V. Zakharevich
    • 1
  • V. N. Danilenko
    • 1
    Email author
  1. 1.Vavilov Institute of General GeneticsRussian Academy of SciencesMoscowRussia
  2. 2.Bach Institute of BiochemistryRussian Academy of SciencesMoscowRussia

Personalised recommendations