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A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme

Author

Listed:
  • Amy E. Hutton

    (The University of Manchester)

  • Jake Foster

    (The University of Manchester)

  • Rebecca Crawshaw

    (The University of Manchester)

  • Florence J. Hardy

    (The University of Manchester)

  • Linus O. Johannissen

    (The University of Manchester)

  • Thomas M. Lister

    (The University of Manchester)

  • Emilie F. Gérard

    (The University of Manchester)

  • Zachary Birch-Price

    (The University of Manchester)

  • Richard Obexer

    (The University of Manchester)

  • Sam Hay

    (The University of Manchester)

  • Anthony P. Green

    (The University of Manchester)

Abstract

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.

Suggested Citation

  • 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.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-46123-z
    DOI: 10.1038/s41467-024-46123-z
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    References listed on IDEAS

    as
    1. 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.
    2. Sarah L. Lovelock & Rebecca Crawshaw & Sophie Basler & Colin Levy & David Baker & Donald Hilvert & Anthony P. Green, 2022. "The road to fully programmable protein catalysis," Nature, Nature, vol. 606(7912), pages 49-58, June.
    3. Ningning Sun & Jianjian Huang & Junyi Qian & Tai-Ping Zhou & Juan Guo & Langyu Tang & Wentao Zhang & Yaming Deng & Weining Zhao & Guojiao Wu & Rong-Zhen Liao & Xi Chen & Fangrui Zhong & Yuzhou Wu, 2022. "Enantioselective [2+2]-cycloadditions with triplet photoenzymes," Nature, Nature, vol. 611(7937), pages 715-720, November.
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