Pyrophosphate-Dependent Enzymes in Methanotrophs: New Findings and Views

  • Valentina N. KhmeleninaEmail author
  • Olga N. Rozova
  • Ilya R. Akberdin
  • Marina G. Kalyuzhnaya
  • Yuri A. Trotsenko


Inorganic pyrophosphate (PPi) is a by-product of cellular anabolic processes whose recycle by inorganic pyrophosphatases is essential for biosynthetic reactions. While the metabolic role of PPi in anaerobic microorganisms is widely acknowledged, the contribution of PPi to central metabolic pathways in aerobic organisms is less understood. In methanotrophs, the enzyme 6-phosphofructokinase (PPi-PFK, EC utilizes PPi as a phosphoryl donor in an energy-consuming stage of Embden-Meyerhof-Parnas glycolysis, i.e., phosphorylation of fructose-6-posphate into fructose-1,6-bisphosphate. Analysis of genomic data shows that the gene encoding PPi-PFK (pfp) is widespread among proteobacterial methanotrophs, while only rarely present in methylotrophs, thus implying a relationship between the PPi-metabolism and methane oxidation. Hence, a number of PPi-linked enzymes have been investigated, including membranous proton-translocating pyrophosphatase, pyruvate-phosphate dikinase, and phosphoenolpyruvate carboxykinase, in an attempt to uncover methanotrophic metabolic mechanisms that help compensate for the high energetic cost of methane oxidation and reset metabolic pathways for efficient C1-assimilation. The remarkable metabolic versatility of PPi reactions in methanotrophs allows them to grow under quite different environmental conditions, including aerobic respiration and semi-anaerobic formaldehyde fermentation.


Inorganic pyrophosphate Pyrophosphatase, 6-phosphofructokinase Pyruvate, phosphate dikinase PPi-type phosphoenolpyruvate carboxykinase H+-translocating membranous pyrophosphatase Methanotrophs Glycolysis Gluconeogenesis 











pyruvate, phosphate dikinase


inorganic pyrophosphate


phosphoenolpyruvate carboxykinase








proton-translocating pyrophosphatase



The work was supported by the Russian Foundation for Basic Research #18-04-00771 and by the US Department of Energy Bioenergy Technologies Office (DOE-BETO under contract No. DE-AC36-08GO28308).


  1. Akberdin IR, Thompson M, Hamilton R, Desai N, Alexande D, Henard CA, Guarnieri MT, Kalyuzhnaya MG (2018) Methane utilization in Methylomicrobium alcaliphilum 20ZR: a systems approach. Sci Rep 8(1):2512. Scholar
  2. Alves AM, Euverink GJ, Hektor HJ et al (1994) Enzymes of glucose and methanol metabolism in the actinomycete Amycolatopsis methanolica. J Bacteriol 176(22):6827–6835CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bapteste E, Moreira D, Philippe H (2003) Rampant horizontal gene transfer and phospho-donor change in the evolution of the phosphofructokinase. Gene 318:185–191CrossRefPubMedGoogle Scholar
  4. Baxter NJ, Hirt RP, Bodrossy L et al (2002) The ribulose-1,5-bisphosphate carboxylase/oxygenase gene cluster of Methylococcus capsulatus (Bath). Arch Microbiol 177:279–289CrossRefPubMedGoogle Scholar
  5. Baykov AA, Malinen AM, Luoto HH et al (2013) Pyrophosphate-fueled Na+ and H+ transport in prokaryotes. Microbiol Mol Biol Rev 77:267–276CrossRefPubMedPubMedCentralGoogle Scholar
  6. Beck DA, McTaggart TL, Setboonsarng U et al (2015) Multiphyletic origins of methylotrophy in Alphaproteobacteria, exemplified by comparative genomics of Lake Washington isolates. Environ Microbiol 17(3):547–554CrossRefPubMedGoogle Scholar
  7. Beschastny AP, Rozova ON, Khmelenina VN et al (2008) Activities of 6-phosphofructokinases and inorganic pyrophosphatase in aerobic methylotrophic bacteria. Mikrobiologiya (Russian) 77(5):713–715Google Scholar
  8. Beschastny AP, Sokolov AP, Khmelenina VN et al (1992) Purification and properties of pyrophosphate-dependent phosphofructokinase of obligate methanotroph Methylomonas methanica. Biochemistry (Moscow) 57:1215–1221Google Scholar
  9. Chi A, Kemp RG (2000) The primordial high energy compound: ATP or inorganic pyrophosphate? J Biol Chem 275:35677–35679CrossRefPubMedGoogle Scholar
  10. Chiba Y, Kamikawa R, Nakada-Tsukui K, Saito-Nakano Y, Nozaki T (2015) Discovery of PPi-type phosphoenolpyruvate carboxykinase genes in eukaryotes and bacteria. J Biol Chem 290(39):23960–23970CrossRefPubMedGoogle Scholar
  11. Eshinimaev BT, Medvedkova KA, Khmelenina VN et al (2004) New thermophilic methanotrophs of the genus Methylocaldum. Mikrobiologiya (Moscow) 73:530–539Google Scholar
  12. Gest H (1972) Energy conversion and generation of reducing power in bacterial photosynthesis. Adv Microb Physiol 7:243–282CrossRefGoogle Scholar
  13. Kalyuzhnaya MG, Yang S, Rozova ON et al (2013) Highly efficient methane biocatalysis revealed in a methanotrophic bacterium. Nat Commun 4:2785CrossRefPubMedGoogle Scholar
  14. Khmelenina VN, Rozova ON, Trotsenko YA (2011) Characterization of the recombinant pyrophosphate-dependent 6-phosphofructokinases from Methylomicrobium alcaliphilum 20Z and Methylococcus capsulatus Bath. Methods Enzymol 495:1–14CrossRefPubMedGoogle Scholar
  15. Kornberg A (1962) On the metabolic significance of phosphorolytic and pyrophosphorolytic reactions. In: Kasha HPB (ed) Horizons in biochemistry. Academic Press, New York, pp 251–264Google Scholar
  16. Lopez-Marques RL, Perez-Castineira JR, Losada M, Serrano A (2004) Differential regulation of soluble and membrane-bound inorganic pyrophosphatases in the photosynthetic bacterium Rhodospirillum rubrum provides insights into pyrophosphate-based stress bioenergetics. J Bacteriol 186(16):5418–5426CrossRefPubMedPubMedCentralGoogle Scholar
  17. Mansurova SE (1989) Inorganic pyrophosphate in mitochondrial metabolism. Biochim Biophys Acta 977:237–247CrossRefPubMedGoogle Scholar
  18. Mertens E (1991) Pyrophosphate-dependent phosphofructokinase, an anaerobic glycolytic enzyme? FEBS Lett 285:1–5CrossRefPubMedGoogle Scholar
  19. Mertens E (1993) ATP versus pyrophosphate: glycolysis revisited in parasitic protists. Parasitol Today 9:122–126CrossRefPubMedGoogle Scholar
  20. Meurice G, Deborde C, Jacob D et al (2004) In silico exploration of the fructose-6-phosphate phosphorylation step in glycolysis: genomic evidence of the coexistence of an atypical ATP-dependent along with a PPi-dependent phosphofructokinase in Propionibacterium freudenreichii subsp. shermanii. In Silico Biol 4:517–528PubMedGoogle Scholar
  21. Murrell JC, Gilbert B, McDonald IR (2000) Molecular biology and regulation of methane monooxygenase. Arch Microbiol 173:325–332CrossRefPubMedGoogle Scholar
  22. O’Brien WE, Bowin S, Wood HG (1975) Isolation and characterization of a pyrophosphate-dependent phosphofructokinase from Propionibacterium shermanii. J Biol Chem 250:8690–8695PubMedGoogle Scholar
  23. Pfleiderer C, Klemme JH (1980) Pyrophosphate-dependent D-fructose-6-phosphate-phosphotransferase in Rhodospirillaceae. Z Naturforsch 35:229–238CrossRefGoogle Scholar
  24. Reeves RE, Serrano R, South DJ (1976) 6-Phosphofructokinase (pyrophosphate). Properties of the enzyme from Entamoeba histolytica and its reaction mechanism. J Biol Chem 251:2958–2962PubMedGoogle Scholar
  25. Reeves RE, South DJ, Blytt HJ et al (1974) Pyrophosphate: d-fructose 6-phosphate 1-phosphotransferase: a new enzyme with the glycolytic function of 6-phosphofructokinase. J Biol Chem 249:7737–7741PubMedGoogle Scholar
  26. Renier A, De Faria SM, Jourand P et al (2011) Nodulation of Crotalaria podocarpa DC by Methylobacterium nodulans displays very unusual features. J Exp Bot 62(10):3693–3697CrossRefPubMedGoogle Scholar
  27. Reshetnikov AS, Rozova ON, Khmelenina VN et al (2008) Сharacterization of pyrophosphate-dependent 6-phosphofructokinase from Methylococcus capsulatus Bath. FEMS Microbiol Lett 288:202–210CrossRefPubMedGoogle Scholar
  28. Rozova ON, Khmelenina VN, Vuilleumier S et al (2010) Characterisation of the recombinant pyrophosphate-dependent 6-phosphofructokinase from halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. Res Microbiol 161:861–868CrossRefPubMedGoogle Scholar
  29. Rozova ON, Khmelenina VN, Trotsenko YA (2012) Characterization of recombinant PPi-dependent 6-phosphofructokinases from Methylosinus trichosporium OB3b and Methylobacterium nodulans ORS 2060. Biochemistry (Mosc) 77(3):288–295CrossRefGoogle Scholar
  30. Slamovits CH, Keeling PJ (2006) Pyruvate-phosphate dikinase of oxymonads and parabasalia and the evolution of pyrophosphate-dependent glycolysis in anaerobic eukaryotes. Eukaryot Cell 5:148–154CrossRefPubMedPubMedCentralGoogle Scholar
  31. Scöcke L, Schink B (1998) Membrane-bound proton-translocating pyrophosphatase of Syntrophus gentianae, a syntrophically benzoate-degrading fermenting bacterium. Eur J Biochem 256:589–594CrossRefGoogle Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Valentina N. Khmelenina
    • 1
    Email author
  • Olga N. Rozova
    • 1
  • Ilya R. Akberdin
    • 2
  • Marina G. Kalyuzhnaya
    • 2
  • Yuri A. Trotsenko
    • 1
  1. 1.G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, RASPushchinoRussia
  2. 2.Department of Biology and Viral Information InstituteSan Diego State UniversitySan DiegoUSA

Personalised recommendations