Advertisement

Applied Microbiology and Biotechnology

, Volume 99, Issue 20, pp 8465–8474 | Cite as

Identification of sucrose synthase in nonphotosynthetic bacteria and characterization of the recombinant enzymes

  • Margo Diricks
  • Frederik De Bruyn
  • Paul Van Daele
  • Maarten Walmagh
  • Tom Desmet
Biotechnologically relevant enzymes and proteins

Abstract

Sucrose synthase (SuSy) catalyzes the reversible conversion of sucrose and a nucleoside diphosphate into fructose and nucleotide (NDP)-glucose. To date, only SuSy’s from plants and cyanobacteria, both photosynthetic organisms, have been characterized. Here, four prokaryotic SuSy enzymes from the nonphotosynthetic organisms Nitrosomonas Europaea (SuSyNe), Acidithiobacillus caldus (SuSyAc), Denitrovibrio acetiphilus (SusyDa), and Melioribacter roseus (SuSyMr) were recombinantly expressed in Escherichia coli and thoroughly characterized. The purified enzymes were found to display high-temperature optima (up to 80 °C), high activities (up to 125 U/mg), and high thermostability (up to 15 min at 60 °C). Furthermore, SuSyAc, SuSyNe, and SuSyDa showed a clear preference for ADP as nucleotide, as opposed to plant SuSy’s which prefer UDP. A structural and mutational analysis was performed to elucidate the difference in NDP preference between eukaryotic and prokaryotic SuSy’s. Finally, the physiological relevance of this enzyme specificity is discussed in the context of metabolic pathways and genomic organization.

Keywords

Sucrose synthase Sucrose metabolism Photosynthesis Nucleotide sugar 

Notes

Acknowledgments

The authors wish to thank the Special Research Fund (BOF) of Ghent University (MRP-project “Ghent Bio-Economy” and PhD-scholarship to MD), as well as the EC (FP7-project “SuSy”, grant agreement no. 613633) for financial support.

Supplementary material

253_2015_6548_MOESM1_ESM.pdf (559 kb)
ESM 1 (PDF 558 kb)

References

  1. Aerts D, Verhaeghe T, De Mey M, Desmet T, Soetaert W (2011) A constitutive expression system for high-throughput screening. Eng Life Sci 11:10–19. doi: 10.1002/elsc.201000065 CrossRefGoogle Scholar
  2. Baroja-Fernández E, Muñoz FJ, Saikusa T, Rodríguez-López M, Akazawa T, Pozueta-Romero J (2003) Sucrose synthase catalyzes the de novo production of ADPglucose linked to starch biosynthesis in heterotrophic tissues of plants. Plant Cell Physiol 44:500–509CrossRefPubMedGoogle Scholar
  3. Baroja-Fernández E, Muñoz FJ, Li J, Bahaji A, Almagro G, Montero M, Etxeberria E, Hidalgo M, Sesma MT, Pozueta-Romero J (2012) Sucrose synthase activity in the sus1/sus2/sus3/sus4 Arabidopsis mutant is sufficient to support normal cellulose and starch production. Proc Natl Acad Sci U S A 109:321–326. doi: 10.1073/pnas.1117099109 PubMedCentralCrossRefPubMedGoogle Scholar
  4. Brinkmann N, Malissard M, Ramuz M, Römer U, Schumacher T, Berger EG, Elling L, Wandrey C, Liese A (2001) Chemo-enzymatic synthesis of the Galili epitope Gal(alpha)(1→3)Galbeta(1→4)GlcNAc on a homogeneously soluble PEG polymer by a multi-enzyme system. Bioorg Med Chem Lett 11:2503–2506CrossRefPubMedGoogle Scholar
  5. Bungaruang L, Gutmann A, Nidetzky B (2013) Leloir Glycosyltransferases and Natural Product Glycosylation: Biocatalytic Synthesis of the C-Glucoside Nothofagin, a Major Antioxidant of Redbush Herbal Tea. Adv Synth Catal 355:2757–2763. doi: 10.1002/adsc.201300251 PubMedCentralCrossRefPubMedGoogle Scholar
  6. But SY, Khmelenina VN, Reshetnikov AS, Trotsenko YA (2013) Bifunctional sucrose phosphate synthase/phosphatase is involved in the sucrose biosynthesis by Methylobacillus flagellatus KT. FEMS Microbiol Lett 347:43–51. doi: 10.1111/1574-6968.12219 CrossRefPubMedGoogle Scholar
  7. Cardini CE, Leloir LF, Chiriboga J (1955) The biosynthesis of sucrose. J Biol Chem 214:149–155PubMedGoogle Scholar
  8. Cerdobbel A, De Winter K, Aerts D, Kuipers R, Joosten H-J, Soetaert W, Desmet T (2011) Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis. Protein Eng Des Sel 24:829–834. doi: 10.1093/protein/gzr042 CrossRefPubMedGoogle Scholar
  9. Chen P-J, Wei T-C, Chang Y-T, Lin L-P (2004) Purification and characterization of carboxymethyl cellulase from Sinorhizobium fredii. Bot Bull Acad Sin 45:111–118Google Scholar
  10. Chua TK, Bujnicki JM, Tan T-C, Huynh F, Patel BK, Sivaraman J (2008) The structure of sucrose phosphate synthase from Halothermothrix orenii reveals its mechanism of action and binding mode. Plant Cell 20:1059–1072. doi: 10.1105/tpc.107.051193 PubMedCentralCrossRefPubMedGoogle Scholar
  11. Copeland R (2000) Enzymes. A practical introduction to structure, mechanism and data analysis. Wiley-VCH, New YorkGoogle Scholar
  12. 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 Physiol 143:1385–1397. doi: 10.1104/pp. 106.091736 PubMedCentralCrossRefPubMedGoogle Scholar
  13. Cumino AC, Perez-Cenci M, Giarrocco LE, Salerno GL (2010) The proteins involved in sucrose synthesis in the marine cyanobacterium Synechococcus sp. PCC 7002 are encoded by two genes transcribed from a gene cluster. FEBS Lett 584:4655–4660. doi: 10.1016/j.febslet.2010.10.040 CrossRefPubMedGoogle Scholar
  14. Curatti L, Porchia AC, Herrera-Estrella L, Salerno GL (2000) A prokaryotic sucrose synthase gene (susA) isolated from a filamentous nitrogen-fixing cyanobacterium encodes a protein similar to those of plants. Planta 211:729–735CrossRefPubMedGoogle Scholar
  15. 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 228:617–625. doi: 10.1007/s00425-008-0764-7 CrossRefPubMedGoogle Scholar
  16. De Bruyn F, Maertens J, Beauprez J, Soetaert W, De Mey M (2015) Biotechnological advances in UDP-sugar based glycosylation of small molecules. Biotechnol Adv. doi: 10.1016/j.biotechadv.2015.02.005 PubMedGoogle Scholar
  17. Delmer DP (1972) The Purification and Properties of Sucrose Synthetase from Etiolated Phaseolus aureus Seedlings. J Biol Chem 247:3822–3828PubMedGoogle Scholar
  18. Elling L, Kula M-R (1995) Characterization of sucrose synthase from rice grains for the enzymatic synthesis of UDP and TDP glucose. Enzym Microb Technol 17:929–934. doi: 10.1016/0141-0229(94)00017-L CrossRefGoogle Scholar
  19. Elling L, Grothus M, Kula MR (1993) Investigation of sucrose synthase from rice for the synthesis of various nucleotide sugars and saccharides. Glycobiology 3:349–355CrossRefPubMedGoogle Scholar
  20. Empadinhas N, da Costa MS (2008) Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes. Int Microbiol 11:151–161PubMedGoogle Scholar
  21. Figueroa CM, Asención Diez 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 587:165–169. doi: 10.1016/j.febslet.2012.11.011 CrossRefPubMedGoogle Scholar
  22. Gibson DG, Young L, Chuang R, Venter JC, Iii CAH, Smith HO, America N (2009) Enzymatic assembly of DNA molecules up to several hundred kilobases. 6:12–16. doi: 10.1038/NMETH.1318
  23. Gutmann A, Bungaruang L, Weber H, Leypold M, Breinbauer R, Nidetzky B (2014) Towards the synthesis of glycosylated dihydrochalcone natural products using glycosyltransferase-catalysed cascade reactions. Green Chem 16:4417–4425. doi: 10.1039/C4GC00960F CrossRefGoogle Scholar
  24. Haigler CH, Ivanova-Datcheva M, Hogan PS, Salnikov VV, Hwang S, Martin K, Delmer DP (2001) Carbon partitioning to cellulose synthesis. Plant Mol Biol 47:29–51CrossRefPubMedGoogle Scholar
  25. Huang DY, Wang AY (1998) Purification and characterization of sucrose synthase isozymes from etiolated rice seedlings. Biochem Mol Biol Int 46:107–113PubMedGoogle Scholar
  26. Jayashree B, Pradeep R, Kumar A, Gopal B (2008) Correlation between the Sucrose Synthase Protein Subfamilies, Variations in Structure and Expression in Stress-derived Expressed Sequence Tag Datasets. J Proteomics Bioinform 01:408–423. doi: 10.4172/jpb.1000050 CrossRefGoogle Scholar
  27. Klotz KL, Finger FL, Shelver WL (2003) Characterization of two sucrose synthase isoforms in sugarbeet root. Plant Physiol Biochem 41:107–115. doi: 10.1016/S0981-9428(02)00024-4 CrossRefGoogle Scholar
  28. Koch K (2004) Sucrose metabolism: regulatory mechanisms and pivotal roles in sugar sensing and plant development. Curr Opin Plant Biol 7:235–246. doi: 10.1016/j.pbi.2004.03.014 CrossRefPubMedGoogle Scholar
  29. Kolman MA, Torres LL, Martin ML, Salerno GL (2012) Sucrose synthase in unicellular cyanobacteria and its relationship with salt and hypoxic stress. Planta 235:955–964. doi: 10.1007/s00425-011-1542-5 CrossRefPubMedGoogle Scholar
  30. Lee JC, Timasheff SN (1981) The stabilization of proteins by sucrose. J Biol Chem 256:7193–7201PubMedGoogle Scholar
  31. Leslie SB, Israeli E, Lighthart B, Crowe JH, Crowe LM (1995) Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol 61:3592–3597PubMedCentralPubMedGoogle Scholar
  32. Lunn JE (2002) Evolution of sucrose synthesis. Plant Physiol 128:1490–1500. doi: 10.1104/pp. 010898 PubMedCentralCrossRefPubMedGoogle Scholar
  33. 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
  34. Mao X, Ma Q, Zhou C, Chen X, Zhang H, Yang J, Mao F, Lai W, Xu Y (2014) DOOR 2.0: presenting operons and their functions through dynamic and integrated views. Nucleic Acids Res 42:D654–D659. doi: 10.1093/nar/gkt1048 PubMedCentralCrossRefPubMedGoogle Scholar
  35. Martínez-Noël GM, Cumino AC, Kolman Mde L, Salerno GL (2013) First evidence of sucrose biosynthesis by single cyanobacterial bimodular proteins. FEBS Lett 587:1669–1674. doi: 10.1016/j.febslet.2013.04.012
  36. Masada S, Kawase Y, Nagatoshi M, Oguchi Y, Terasaka K, Mizukami H (2007) An efficient chemoenzymatic production of small molecule glucosides with in situ UDP-glucose recycling. FEBS Lett 581:2562–2566. doi: 10.1016/j.febslet.2007.04.074 CrossRefPubMedGoogle Scholar
  37. Morell M, Copeland L (1985) Sucrose synthase of soybean nodules. Plant Physiol 78:149–154PubMedCentralCrossRefPubMedGoogle Scholar
  38. Porchia AC, Salerno GL (1996) Sucrose biosynthesis in a prokaryotic organism: Presence of two sucrose-phosphate synthases in Anabaena with remarkable differences compared with the plant enzymes. Proc Natl Acad Sci U S A 93:13600–13604PubMedCentralCrossRefPubMedGoogle Scholar
  39. 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
  40. Reed R (1986) Organic solute accumulation in osmotically stressed cyanobacteria. FEMS Microbiol Lett 39:51–56. doi: 10.1016/0378-1097(86)90060-1 CrossRefGoogle Scholar
  41. Reid SJ, Abratt VR (2005) Sucrose utilisation in bacteria: genetic organisation and regulation. Appl Microbiol Biotechnol 67:312–321. doi: 10.1007/s00253-004-1885-y CrossRefPubMedGoogle Scholar
  42. Roby C, Cortès S, Gromova M, Le Bail J-L, Roberts JKM (2002) Sucrose cycling in heterotrophic plant cell metabolism: first step towards an experimental model. Mol Biol Rep 29:145–149CrossRefPubMedGoogle Scholar
  43. Ross HA, Davies HV (1992) Purification and Characterization of Sucrose Synthase from the Cotyledons of Vicia faba L. Plant Physiol 100:1008–1013PubMedCentralCrossRefPubMedGoogle Scholar
  44. Salerno GL, Curatti L (2003) Origin of sucrose metabolism in higher plants: when, how and why? Trends Plant Sci 8:63–69. doi: 10.1016/S1360-1385(02)00029-8 CrossRefPubMedGoogle Scholar
  45. Sanchis J, Fernández L, Carballeira JD, Drone J, Gumulya Y, Höbenreich H, Kahakeaw D, Kille S, Lohmer R, Peyralans JJ-P, Podtetenieff J, Prasad S, Soni P, Taglieber A, Wu S, Zilly FE, Reetz MT (2008) Improved PCR method for the creation of saturation mutagenesis libraries in directed evolution: application to difficult-to-amplify templates. Appl Microbiol Biotechnol 81:387–397. doi: 10.1007/s00253-008-1678-9 CrossRefPubMedGoogle Scholar
  46. Sebková V, Unger C, Hardegger M, Sturm A (1995) Biochemical, physiological, and molecular characterization of sucrose synthase from Daucus carota. Plant Physiol 108:75–83PubMedCentralCrossRefPubMedGoogle Scholar
  47. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Söding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539. doi: 10.1038/msb.2011.75 PubMedCentralCrossRefPubMedGoogle Scholar
  48. Son MH, Kim B-G, Kim DH, Jin M, Kim K, Ahn J-H (2009) Production of flavonoid o-glucoside using sucrose synthase and flavonoid o-glucosyltransferase fusion protein. J Microbiol Biotechnol 19:709–712PubMedGoogle Scholar
  49. Subbaiah CC, Palaniappan A, Duncan K, Rhoads DM, Huber SC, Sachs MM (2006) Mitochondrial localization and putative signaling function of sucrose synthase in maize. J Biol Chem 281:15625–15635. doi: 10.1074/jbc.M600355200 CrossRefPubMedGoogle Scholar
  50. Taboada B, Ciria R, Martinez-Guerrero CE, Merino E (2012) ProOpDB: prokaryotic operon database. Nucleic Acids Res 40:D627–D631. doi: 10.1093/nar/gkr1020 PubMedCentralCrossRefPubMedGoogle Scholar
  51. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729. doi: 10.1093/molbev/mst197 PubMedCentralCrossRefPubMedGoogle Scholar
  52. Tanase K, Yamaki S (2000) Purification and characterization of two sucrose synthase isoforms from Japanese pear fruit. Plant Cell Physiol 41:408–414CrossRefPubMedGoogle Scholar
  53. Terasaka K, Mizutani Y, Nagatsu A, Mizukami H (2012) In situ UDP-glucose regeneration unravels diverse functions of plant secondary product glycosyltransferases. FEBS Lett 586:4344–4350. doi: 10.1016/j.febslet.2012.10.045 CrossRefPubMedGoogle Scholar
  54. Tsai C-Y (1974) Sucrose-udp glucosyltransferase of Zea mays endosperm. Phytochemistry 13:885–891. doi: 10.1016/S0031-9422(00)91418-3 CrossRefGoogle Scholar
  55. Vargas W, Cumino A, Salerno GL (2003) Cyanobacterial alkaline/neutral invertases. Origin of sucrose hydrolysis in the plant cytosol? Planta 216:951–960. doi: 10.1007/s00425-002-0943-x PubMedGoogle Scholar
  56. Waffenschmidt S, Jaenicke L (1987) Assay of Reducing Sugars in the Nanomole Range with 2, 2 ’ -Bicinchoninate. Anal Biochem 165:337–340CrossRefPubMedGoogle Scholar
  57. Winter H, Huber SC (2000) Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes. Crit Rev Biochem Mol Biol 35:253–289. doi: 10.1080/10409230008984165 CrossRefPubMedGoogle Scholar
  58. Zervosen A, Römer U, Elling L (1998) Application of recombinant sucrose synthase-large scale synthesis of ADP-glucose. J Mol Catal B Enzym 5:25–28. doi: 10.1016/S1381-1177(98)00040-X CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Margo Diricks
    • 1
  • Frederik De Bruyn
    • 1
  • Paul Van Daele
    • 1
  • Maarten Walmagh
    • 1
  • Tom Desmet
    • 1
  1. 1.Centre for Industrial Biotechnology and Biocatalysis, Department of Biochemical and Microbial TechnologyGhent UniversityGhentBelgium

Personalised recommendations