, Volume 22, Issue 3, pp 433–445 | Cite as

The genes and enzymes of sucrose metabolism in moderately thermophilic methanotroph Methylocaldum szegediense O12

  • Sergey Y. But
  • Natalia P. Solntseva
  • Svetlana V. Egorova
  • Ildar I. Mustakhimov
  • Valentina N. Khmelenina
  • Alexander Reshetnikov
  • Yuri A. Trotsenko
Original Paper


Four enzymes involved in sucrose metabolism: sucrose phosphate synthase (Sps), sucrose phosphate phosphatase (Spp), sucrose synthase (Sus) and fructokinase (FruK), were obtained as his-tagged proteins from the moderately thermophilic methanotroph Methylocaldum szegediense O12. Sps, Spp, FruK and Sus demonstrated biochemical properties similar to those of other bacterial counterparts, but the translated amino acid sequences of Sps and Spp displayed high divergence from the respective microbial enzymes. The Sus of M. szegediense O12 catalyzed the reversible reaction of sucrose cleavage in the presence of ADP or UDP and preferred ADP as a substrate, thus implying a connection between sucrose and glycogen metabolism. Sus-like genes were found only in a few methanotrophs, whereas amylosucrase was generally used in sucrose cleavage in this group of bacteria. Like other microbial fructokinases, FruK of M. szegediense O12 showed a high specificity to fructose.


Methylocaldum szegediense Sucrose phosphate synthase Sucrose phosphate phosphatase Sucrose synthase Fructokinase 



The work was supported by the Russian Foundation for Basic Research #16-04-00462-a and by the Russian Scientific Foundation #18-14-00326. The authors are grateful to all members of the Organization for Methanotroph Genome Analysis for their collaboration (OMeGA), the U.S. Department of Energy Joint Genome Institute and Genoscope for the access to methanotrophic genomes for comparative analyses.

Supplementary material

792_2018_1006_MOESM1_ESM.pdf (1.8 mb)
Supplementary material 1 (PDF 1878 kb)


  1. Akutsu J, Zhang Z, Morita R, Kawarabayasi Y (2015) Identification and characterization of a thermostable bifunctional enzyme with phosphomannose isomerase and sugar-1-phosphate nucleotidylyltransferase activities from a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Extremophiles 19(6):1077–1085CrossRefPubMedGoogle Scholar
  2. Bodrossy L, Holmes EM, Holmes AJ, Kovacs KL, Murrell JC (1997) Analysis of 16S rRNA and methane monooxygenase gene sequences reveals a novel group of thermotolerant and thermophilic methanotrophs, Methylocaldumgen. nov. Arch Microbiol 168:493–503CrossRefPubMedGoogle Scholar
  3. Bruneau JM, Worrell AC, Cambou B, Lando D, Voelker TA (1991) Sucrose phosphate synthase, a key enzyme for sucrose biosynthesis in plants. Plant Physiol 96:473–478CrossRefPubMedPubMedCentralGoogle Scholar
  4. But SY, Rozova ON, Khmelenina VN, Reshetnikov AS, Trotsenko YA (2012) Properties of recombinant ATP dependent fructokinase from the halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. Biochemistry (Moscow) 77:372–377CrossRefGoogle Scholar
  5. But SY, Khmelenina VN, Reshetnikov AS, Trotsenko YA (2013a) Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT. FEMS Microbiol Lett 347:43–51CrossRefPubMedGoogle Scholar
  6. But SY, Khmelenina VN, Reshetnikov AS, Trotsenko YA (2013b) Construction and characterization of Methylomicrobium alcaliphilum 20Z knockout mutants defective in sucrose and ectoine biosynthesis genes. Microbiology (Moscow) 82(2):253–255CrossRefGoogle Scholar
  7. But SY, Khmelenina VN, Reshetnikov AS, Mustakhimov II, Kalyuzhnaya MG, Trotsenko YA (2015) Sucrose metabolism in halotolerant methanotroph Methylomicrobium alcaliphilum 20Z. Arch Microbiol 197(3):471–480CrossRefPubMedGoogle Scholar
  8. Caescu C, Vidal O, Krzewinski O, Artenie V, Bouquelet S (2004) Bifidobacterium longum requires a fructokinase (Frk; ATP:d-fructose-6-phosphotransferase, EC for fructose catabolism. J Bacteriol 186:6515–6525CrossRefPubMedPubMedCentralGoogle Scholar
  9. Cumino A, Ekeroth C, Salerno GL (2001) Sucrose-phosphate phosphatase from Anabaena sp. strain PCC 7120: isolation of the protein and gene revealed significant structural differences from the higher-plant enzyme. Planta 214(2):250–256CrossRefPubMedGoogle Scholar
  10. Cumino AC, Marcozzi C, Barreiro R, Salerno GL (2007) Carbon cycling in Anabaena sp. PCC 7120. Sucrose synthesis in the heterocysts and possible role in nitrogen fixation. Plant Phys 143:1385–1397CrossRefGoogle Scholar
  11. Curatti L, Desplats EFP, Abratti G, Limones V, Herrera-Estrella L, Salerno G (1998) Sucrose-phosphate synthase from Synechocystis sp. PCC 6803: identification of the spsA gene and characterization of the enzyme expressed in Escherichia coli. J Bacteriol 180:6776–6779PubMedPubMedCentralGoogle Scholar
  12. Curatti L, Giarrocco LE, Cumino AC, Salerno GL (2008) Sucrose synthase is involved in the conversion of sucrose to polysaccharides in filamentous nitrogen-fixing cyanobacteria. Planta 228:617–625CrossRefPubMedGoogle Scholar
  13. Diricks M, De Bruyn F, Van Daele P, Walmagh M, Desmet T (2015) Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes. Appl Microbiol Biotechnol 99:8465–8474CrossRefPubMedGoogle Scholar
  14. Ehira S, Kimura S, Miyazaki S, Ohmori M (2014) Sucrose synthesis in the nitrogen-fixing Cyanobacterium Anabaena sp. strain PCC 7120 is controlled by the two-component response regulator OrrA. Appl Environ Microbiol 80:5672–5679CrossRefPubMedPubMedCentralGoogle Scholar
  15. Eshinimaev BTS, Medvedkova KA, Khmelenina VN, Suzina NE, Osipov GA, Lysenko AM, TrotsenkoIu A (2004) New thermophilic methanotrophs of the genus Methylocaldum. Mikrobiologiia (Moscow) 73:530–539Google Scholar
  16. Figueroa CM, AsenciónDiez MD, Kuhn ML, McEwen S, Salerno GL, Iglesias AA, Ballicora MA (2013) The unique nucleotide specificity of the sucrose synthase from Thermosynechococcus elongatus. FEBS Lett 587:165–169CrossRefPubMedGoogle Scholar
  17. Islam T, Larsen Ø, Torsvik V, Øvreås L, Panosyan H, Murrell JC, Birkeland N-K, Bodrossy L (2015) Novel methanotrophs of the family Methylococcaceae from different geographical regions and habitats. Microorganisms 3:484–499CrossRefPubMedPubMedCentralGoogle Scholar
  18. Khmelenina VN, Kalyuzhnaya MG, Sakharovsky VG, Suzina NE, Trotsenko YA, Gottschalk G (1999) Osmoadaptation in halophilic and alkaliphilicmethanotrophs. Arch Microbiol 172:321–329CrossRefPubMedGoogle Scholar
  19. King K, Phan P, Rellos P, Scopes RK (1996) Overexpression, purification, and generation of a thermostable variant of Zymomonas mobilis fructokinase. Protein Expr Purif 7:373–376CrossRefPubMedGoogle Scholar
  20. Klähn S, Hagemann M (2011) Compatible solute biosynthesis in cyanobacteria. Environ Microbiol 13:551–562CrossRefPubMedGoogle Scholar
  21. Klotz KL, Finger FL, Shelver WL (2003) Characterization of two sucrose synthase isoforms in sugar beet root. Plant Physiol Biochem 41:107–115CrossRefGoogle Scholar
  22. Kolman MA, Torres LL, Martin ML, Salerno GL (2012) Sucrose synthase in unicellular cyanobacteria and its relationship with salt and hypoxic stress. Planta 235:955–964CrossRefPubMedGoogle Scholar
  23. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedGoogle Scholar
  24. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(− Delta Delta C(T)) method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  25. Lunn J (2002) Evolution of sucrose synthesis Plant Physiol 128:1490–1500PubMedGoogle Scholar
  26. Lunn JE, Price GD, Furbank RT (1999) Cloning and expression of a prokaryotic sucrose-phosphate synthase gene from the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 40:297–305CrossRefPubMedGoogle Scholar
  27. Martínez-Noël GM, Cumino AC, KolmanMde L, Salerno GL (2013) First evidence of sucrose biosynthesis by single cyanobacterial bimodular proteins. FEBS Lett 587:1669–1674CrossRefPubMedGoogle Scholar
  28. Medvedkova KA, Khmelenina VN, Trotsenko YA (2007) Sucrose as a factor of thermal adaptation of the thermophilic methanotroph Methylocaldum szegediense O-12. Mikrobiologiya (Moscow) 76:500–502CrossRefGoogle Scholar
  29. Medvedkova KA, Khmelenina VN, Baskunov BP, TrotsenkoIu A (2008) Synthesis of melanine by a moderately thermophilic methanotroph Methylocaldum szegediense O-12 depends on cultivation temperature. Mikrobiologiya (Moscow) 77:126–128Google Scholar
  30. Murao S, Nakatani A, Kaneda N (1995) Isolation and characterization of fructokinase from Pseudomonas sp. KN-21. Biosci Biotechnol Biochem 59:1798–1800CrossRefGoogle Scholar
  31. Porchia AC, Curatti L, Salerno GL (1999) Sucrose metabolism in cyanobacteria: sucrose synthase from Anabaena sp. strain PCC 7119 is remarkably different from the plant enzymes with respect to substrate affinity and amino-terminal sequence. Planta 210:34–40CrossRefPubMedGoogle Scholar
  32. Reshetnikov AS, Khmelenina VN, Trotsenko YA (2006) Characterization of the ectoine biosynthesis genes of haloalkalotolerant obligate methanotroph “Methylomicrobium alcaliphilum 20Z”. Arch Microbiol 184:286–297CrossRefPubMedGoogle Scholar
  33. Rozova ON, Khmelenina VN, Vuilleumier S, Trotsenko YA (2010) Characterization of recombinant pyrophosphate-dependent 6-phosphofructokinase from halotolerant methanotroph Methylomicrobium alcaliphilum20Z. Res Microbiol 161:861–868CrossRefPubMedGoogle Scholar
  34. Salerno GL, Curatti L (2003) Origin of sucrose metabolism in higher plants: when, how and why? Trends Plant Sci 8:63–69CrossRefPubMedGoogle Scholar
  35. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, New YorkGoogle Scholar
  36. Sinha AK, Pathre U, Sane PV (1997) Purification and characterization of sucrose-phosphate synthase from Prosopis juliflora. Phytochemistry 46:441–447CrossRefGoogle Scholar
  37. Slater GG (1969) Stable pattern formation and determination of molecular size by pore-limit electrophoresis. Anal Chem 41(8):1039–1041CrossRefPubMedGoogle Scholar
  38. Thomson JD, Gibson TJ, Plewniak Jeanmougin F, Higgins DG (1997) The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res 24:4876–4882CrossRefGoogle Scholar
  39. Torres LL, Salerno GL (2007) A metabolic pathway leading to mannosylfructose biosynthesis in Agrobacterium tumefaciens uncovers a family of mannosyltransferases. Proc Natl Acad Sci USA 104:14318–14323CrossRefPubMedPubMedCentralGoogle Scholar
  40. Winter H, Huber S (2000) Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Crit Rev Plant Sci 19:31–67CrossRefGoogle Scholar
  41. Wu B, Zhang Y, Zheng R, Guo C, Wang PG (2002) Bifunctional phosphomannose isomerase/GDP-d-mannose pyrophosphorylase is the point of control for GDP-d-mannose biosynthesis in Helicobacter pylori. FEBS Lett 519:87–92CrossRefPubMedGoogle Scholar
  42. Yang Y, Shan J, Zhang J, Zhang X, Xie S, Liu Y (2014) Ammonia- and methane-oxidizing microorganisms in high-altitude wetland sediments and adjacent agricultural soils. Appl Microbiol Biotechnol 98:10197–10209CrossRefPubMedGoogle Scholar
  43. Yen SF, Su JC, Sung HY (1994) Purification and characterization of rice sucrose synthase isozymes. Biochem Mol Biol Int 34:613–620PubMedGoogle Scholar

Copyright information

© Springer Japan KK, part of Springer Nature 2018

Authors and Affiliations

  • Sergey Y. But
    • 1
  • Natalia P. Solntseva
    • 1
    • 2
  • Svetlana V. Egorova
    • 1
    • 2
  • Ildar I. Mustakhimov
    • 1
  • Valentina N. Khmelenina
    • 1
  • Alexander Reshetnikov
    • 1
  • Yuri A. Trotsenko
    • 1
  1. 1.Laboratory of Methylotrophy, Skryabin Institute of Biochemistry and Physiology of MicroorganismsRussian Academy of SciencesPushchinoRussia
  2. 2.Pushchino State Institute of Natural SciencesPushchinoRussia

Personalised recommendations