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Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm

Nature Nanotechnology volume 9, pages 531–536 (2014)Cite this article

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Abstract

Swinging arms are a key functional component of multistep catalytic transformations in many naturally occurring multi-enzyme complexes1. This arm is typically a prosthetic chemical group that is covalently attached to the enzyme complex via a flexible linker, allowing the direct transfer of substrate molecules between multiple active sites within the complex2,3,4. Mimicking this method of substrate channelling outside the cellular environment requires precise control over the spatial parameters of the individual components within the assembled complex. DNA nanostructures can be used to organize functional molecules with nanoscale precision5,6,7 and can also provide nanomechanical control8,9,10,11. Until now, protein–DNA assemblies12 have been used to organize cascades of enzymatic reactions by controlling the relative distance and orientation of enzymatic components13,14,15,16 or by facilitating the interface between enzymes/cofactors and electrode surfaces17,18. Here, we show that a DNA nanostructure can be used to create a multi-enzyme complex in which an artificial swinging arm facilitates hydride transfer between two coupled dehydrogenases. By exploiting the programmability of DNA nanostructures, key parameters including position, stoichiometry and inter-enzyme distance can be manipulated for optimal activity.

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Figure 1: Design and characterization of an NAD+-modified swinging arm providing restricted diffusion of NAD+/NADH between two dehydrogenases.
Figure 2: Characterization of local diffusive transport by the poly(T)20 swinging arm.
Figure 3: Characterization of enzymatic activity in the G6pDH–NAD+–MDH swinging-arm structures.
Figure 4: Specificity of the G6pDH–NAD+–MDH swinging-arm structure when free LDH is introduced to compete for the NADH produced by G6pDH.

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Acknowledgements

This work was supported by an Army Research Office MURI award (no. W911NF-12-1-0420 to H.Y., N.G.W. and N.W.W.), a National Science Foundation grant (no. 1033222 to N.W.W. and H.Y.) and an Army Research Office grant (no. W911NF-11-1-0137 to H.Y. and Y.L.). H.Y. is supported by the Presidential Strategic Initiative Fund from Arizona State University. The authors are grateful to J. Nangreave for her help in editing the manuscript and W. Li for his assistance with the FastScan AFM.

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Author notes

  1. Jinglin Fu

    Present address: Present address: Department of Chemistry, Center for Computational and Integrative Biology, Rutgers University at Camden, Camden, New Jersey 08102, USA,

  2. Jinglin Fu and Yuhe Renee Yang: These authors contributed equally to this work

Authors and Affiliations

  1. Center for Molecular Design and Biomimicry, Biodesign Institute at Arizona State University, Tempe, 85287, Arizona, USA

    Jinglin Fu, Yuhe Renee Yang, Minghui Liu, Yan Liu & Hao Yan

  2. Center for Innovations in Medicine, Biodesign Institute at Arizona State University, Tempe, 85287, Arizona, USA

    Jinglin Fu & Neal W. Woodbury

  3. Department of Chemistry and Biochemistry, Arizona State University, Tempe, 85287, Arizona, USA

    Yuhe Renee Yang, Minghui Liu, Yan Liu, Neal W. Woodbury & Hao Yan

  4. Department of Chemistry, Single Molecule Analysis Group, University of Michigan at Ann Arbor, Ann Arbor, 48109, Michigan, USA

    Alexander Johnson-Buck & Nils G. Walter

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Contributions

J.F., H.Y. and N.W.W. conceived the concepts. J.F. and Y.Y. designed DNA nanostructures, performed the enzyme–DNA structure assembly and activity assay, and analysed data. A.J-B. performed the smFRET experiments and analysed data. M.L performed the 4 × 4 enzyme structures experiments. J.F., A.J-B. and Y.Y. wrote the manuscript. H.Y., N.W.W., Y.L. and N.G.W. discussed the results and commented on the manuscript.

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Correspondence to Jinglin Fu or Hao Yan.

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Fu, J., Yang, Y., Johnson-Buck, A. et al. Multi-enzyme complexes on DNA scaffolds capable of substrate channelling with an artificial swinging arm. Nature Nanotech 9, 531–536 (2014). https://doi.org/10.1038/nnano.2014.100

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