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Design and evolution of an enzyme with a non-canonical organocatalytic mechanism

Author

Listed:
  • Ashleigh J. Burke

    (University of Manchester)

  • Sarah L. Lovelock

    (University of Manchester)

  • Amina Frese

    (University of Manchester)

  • Rebecca Crawshaw

    (University of Manchester)

  • Mary Ortmayer

    (University of Manchester)

  • Mark Dunstan

    (University of Manchester)

  • Colin Levy

    (University of Manchester)

  • Anthony P. Green

    (University of Manchester)

Abstract

The combination of computational design and laboratory evolution is a powerful and potentially versatile strategy for the development of enzymes with new functions1–4. However, the limited functionality presented by the genetic code restricts the range of catalytic mechanisms that are accessible in designed active sites. Inspired by mechanistic strategies from small-molecule organocatalysis5, here we report the generation of a hydrolytic enzyme that uses Nδ-methylhistidine as a non-canonical catalytic nucleophile. Histidine methylation is essential for catalytic function because it prevents the formation of unreactive acyl-enzyme intermediates, which has been a long-standing challenge when using canonical nucleophiles in enzyme design6–10. Enzyme performance was optimized using directed evolution protocols adapted to an expanded genetic code, affording a biocatalyst capable of accelerating ester hydrolysis with greater than 9,000-fold increased efficiency over free Nδ-methylhistidine in solution. Crystallographic snapshots along the evolutionary trajectory highlight the catalytic devices that are responsible for this increase in efficiency. Nδ-methylhistidine can be considered to be a genetically encodable surrogate of the widely employed nucleophilic catalyst dimethylaminopyridine11, and its use will create opportunities to design and engineer enzymes for a wealth of valuable chemical transformations.

Suggested Citation

  • Ashleigh J. Burke & Sarah L. Lovelock & Amina Frese & Rebecca Crawshaw & Mary Ortmayer & Mark Dunstan & Colin Levy & Anthony P. Green, 2019. "Design and evolution of an enzyme with a non-canonical organocatalytic mechanism," Nature, Nature, vol. 570(7760), pages 219-223, June.
  • Handle: RePEc:nat:nature:v:570:y:2019:i:7760:d:10.1038_s41586-019-1262-8
    DOI: 10.1038/s41586-019-1262-8
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    Cited by:

    1. Kaiyuan Wang & Qing Hong & Caixia Zhu & Yuan Xu & Wang Li & Ying Wang & Wenhao Chen & Xiang Gu & Xinghua Chen & Yanfeng Fang & Yanfei Shen & Songqin Liu & Yuanjian Zhang, 2024. "Metal-ligand dual-site single-atom nanozyme mimicking urate oxidase with high substrates specificity," Nature Communications, Nature, vol. 15(1), pages 1-12, December.
    2. Amy E. Hutton & Jake Foster & Rebecca Crawshaw & Florence J. Hardy & Linus O. Johannissen & Thomas M. Lister & Emilie F. Gérard & Zachary Birch-Price & Richard Obexer & Sam Hay & Anthony P. Green, 2024. "A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme," Nature Communications, Nature, vol. 15(1), pages 1-13, December.
    3. Haoran Huang & Tao Yan & Chang Liu & Yuxiang Lu & Zhigang Wu & Xingchu Wang & Jie Wang, 2024. "Genetically encoded Nδ-vinyl histidine for the evolution of enzyme catalytic center," Nature Communications, Nature, vol. 15(1), pages 1-11, December.

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