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A water-soluble DsbB variant that catalyzes disulfide-bond formation in vivo

Nature Chemical Biology volume 13, pages 10221028 (2017) | Download Citation

Abstract

Escherichia coli DsbB is a transmembrane enzyme that catalyzes the reoxidation of the periplasmic oxidase DsbA by ubiquinone. Here, we sought to convert membrane-bound DsbB into a water-soluble biocatalyst by leveraging a previously described method for in vivo solubilization of integral membrane proteins (IMPs). When solubilized DsbB variants were coexpressed with an export-defective copy of DsbA in the cytoplasm of wild-type E. coli cells, artificial oxidation pathways were created that efficiently catalyzed de novo disulfide-bond formation in a range of substrate proteins, in a manner dependent on both DsbA and quinone. Hence, DsbB solubilization was achieved with preservation of both catalytic activity and substrate specificity. Moreover, given the generality of the solubilization technique, the results presented here should pave the way to unlocking the biocatalytic potential of other membrane-bound enzymes whose utility has been limited by poor stability of IMPs outside of their native lipid-bilayer context.

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Acknowledgements

We thank L. Ruddock (University of Oulu) for providing plasmids used in this study. We thank C. Sevier (Cornell University) for helpful discussions regarding the manuscript. This work is based on research conducted at the Cornell High Energy Synchrotron Source (CHESS), which is supported by the NSF and the NIH/NIGMS under NSF award DMR-1332208, in the Macromolecular Diffraction at CHESS (MacCHESS) facility, which is supported by award GM-103485 from the NIH/NIGMS. This work also made use of the Cornell Center for Materials Research Shared Facilities, which are supported through the NSF MRSEC program (grant no. DMR-1120296). This work is based on work supported by NIH grants R21DA031409-01 (to M.P.D.), NSF grants CBET 1159581 and CBET 1264701 (both to M.P.D.), a Ford Foundation Predoctoral Fellowship (to M.-P.R.), and a National Science Foundation Graduate Research Fellowship (to M.-P.R.).

Author information

Affiliations

  1. School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York, USA.

    • Dario Mizrachi
    • , Michael-Paul Robinson
    •  & Matthew P DeLisa
  2. New England Biolabs, Ipswich, Massachusetts, USA.

    • Guoping Ren
    • , Na Ke
    •  & Mehmet Berkmen

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Contributions

D.M. designed research, performed all research, analyzed all data and wrote the paper. M.-P.R. performed experiments and analyzed data related to antibody expression. G.R. and N.K. performed experiments and analyzed data related to AMS alkylation, quinone-deficient cPhoA activity, and c-uPA activity. M.B. designed research and analyzed data. M.P.D. designed research, analyzed data, and wrote the paper.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Matthew P DeLisa.

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    Supplementary Results, Supplementary Table 1 and Supplementary Figures 1–11

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DOI

https://doi.org/10.1038/nchembio.2409

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