Biotechnology Letters

, Volume 40, Issue 4, pp 667–672 | Cite as

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction

Original Research Paper
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Abstract

Objective

We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions.

Results

Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes.

Conclusion

A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA–enzyme complexes.

Keywords

A*-tag DNA scaffold Enzyme cycling reaction Firefly luciferase Multi-enzyme co-localization Pyruvate orthophosphate dikinase Proximity effect 

Notes

Acknowledgements

This work was financially supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Kikkoman Corporation for supplying BL assay reagents and PPDK gene.

Supporting information

Supplementary Table 1—DNA sequences in this study.

Supplementary material

10529_2018_2517_MOESM1_ESM.docx (18 kb)
Supplementary material 1 (DOCX 17 kb)

References

  1. Akter F, Mie M, Grimm S, Nygren PA, Kobatake E (2012) Detection of antigens using a protein-DNA chimera developed by enzymatic covalent bonding with phiX Gene A. Anal Chem 84:5040–5046CrossRefPubMedGoogle Scholar
  2. Arakawa H et al (2008) Development of bioluminescent pyrophosphate assay using pyruvate phosphate dikinase and its application to single-nucleotide polymorphism analysis. Anal Biochem 379:86–90CrossRefPubMedGoogle Scholar
  3. Erkelenz M, Kuo CH, Niemeyer CM (2011) DNA-mediated assembly of cytochrome P450 BM3 subdomains. J Am Chem Soc 133:16111–16118CrossRefPubMedGoogle Scholar
  4. Fu JL, Liu MH, Liu Y, Woodbury NW, Yan H (2012) Interenzyme substrate diffusion for an enzyme cascade organized on spatially addressable DNA nanostructures. J Am Chem Soc 134:5516–5519CrossRefPubMedPubMedCentralGoogle Scholar
  5. Funabashi H, Yanagi S, Suzuki S, Mie M, Kobatake E (2015) Assembly of zinc finger motif-fused enzymes on a dsDNA scaffold for catalyzing consecutive reactions with a proximity effect. Biotechnol Lett 37:109–114CrossRefPubMedGoogle Scholar
  6. Kitaoka M et al (2011) Transglutaminase-mediated synthesis of a DNA-(Enzyme)(n) probe for highly sensitive DNA detection. Chem Eur J 17:5387–5392CrossRefPubMedGoogle Scholar
  7. Mashimo Y, Maeda H, Mie M, Kobatake E (2012) Construction of semisynthetic DNA-protein conjugates with Phi X174 Gene-A* protein. Bioconjugate Chem 23:1349–1355CrossRefGoogle Scholar
  8. Newman JD, Setford SJ (2006) Enzymatic biosensors. Mol Biotechnol 32:249–268CrossRefPubMedGoogle Scholar
  9. Niemeyer CM, Koehler J, Wuerdemann C (2002) DNA-directed assembly of bienzymic complexes from in vivo biotinylated NAD(P)H: FMN oxidoreductase and luciferase. ChemBioChem 3:242–245CrossRefPubMedGoogle Scholar
  10. Nojima T, Konno H, Kodera N, Seio K, Taguchi H, Yoshida M (2012) Nano-scale alignment of proteins on a flexible DNA backbone. PLoS ONE 7:e52534CrossRefPubMedPubMedCentralGoogle Scholar
  11. Rajendran A, Nakata E, Nakano S, Morii T (2017) Nucleic-acid-templated enzyme cascades. ChemBioChem 18:696–716CrossRefPubMedGoogle Scholar
  12. Ricca E, Brucher B, Schrittwieser JH (2011) Multi-enzymatic cascade reactions: overview and perspectives. Adv Synth Catal 353:2239–2262CrossRefGoogle Scholar
  13. Sakakibara T, Murakami S, Eisaki N, Nakajima MO, Imai K (1999) An enzymatic cycling method using pyruvate orthophosphate dikinase and firefly luciferase for the simultaneous determination of ATP and AMP (RNA). Anal Biochem 268:94–101CrossRefPubMedGoogle Scholar
  14. Srere PA (1987) Complexes of sequential metabolic enzymes. Annu Rev Biochem 56:89–124CrossRefPubMedGoogle Scholar
  15. Srere PA, Mattiasson B, Mosbach K (1973) An immobilized three-enzyme system: a model for microenvironmental compartmentation in mitochondria. Proc Nat Acad Sci USA 70:2534–2538CrossRefPubMedPubMedCentralGoogle Scholar
  16. Wang SZ, Zhang YH, Ren H, Wang YL, Jiang W, Fang BS (2017) Strategies and perspectives of assembling multi-enzyme systems. Crit Rev Biotechnol 37:1024–1037CrossRefPubMedGoogle Scholar
  17. Wheeldon I, Minteer SD, Banta S, Barton SC, Atanassov P, Sigman M (2016) Substrate channelling as an approach to cascade reactions. Nat Chem 8:299–309CrossRefPubMedGoogle Scholar
  18. Zhou G, Kajiyama T, Gotou M, Kishimoto A, Suzuki S, Kambara H (2006) Enzyme system for improving the detection limit in pyrosequencing. Anal Chem 78:4482–4489CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Life Science and Technology, School of Life Science and TechnologyTokyo Institute of TechnologyYokohama-ShiJapan

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