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Mechanistic conformational and substrate selectivity profiles emerging in the evolution of enzymes via parallel trajectories

Author

Listed:
  • Christos S. Karamitros

    (University of Texas at Austin (UT Austin)
    Nestlé Health Science)

  • Kyle Murray

    (The University of Texas at Dallas
    Inc. Boston)

  • Yoichi Kumada

    (Kyoto Institute of Technology)

  • Kenneth A. Johnson

    (University of Texas at Austin (UT Austin))

  • Sheena D’Arcy

    (The University of Texas at Dallas)

  • George Georgiou

    (University of Texas at Austin (UT Austin)
    University of Texas at Austin (UT Austin)
    University of Texas at Austin (UT Austin)
    The University of Texas at Austin (UT Austin))

Abstract

Laboratory evolution studies have demonstrated that parallel evolutionary trajectories can lead to genetically distinct enzymes with high activity towards a non-preferred substrate. However, it is unknown whether such enzymes have convergent conformational dynamics and mechanistic features. To address this question, we use as a model the wild-type Homo sapiens kynureninase (HsKYNase), which is of great interest for cancer immunotherapy. Earlier, we isolated HsKYNase_66 through an unusual evolutionary trajectory, having a 410-fold increase in the kcat/KM for kynurenine (KYN) and reverse substrate selectivity relative to HsKYNase. Here, by following a different evolutionary trajectory we generate a genetically distinct variant, HsKYNase_93D9, that exhibits KYN catalytic activity comparable to that of HsKYNase_66, but instead it is a “generalist” that accepts 3’-hydroxykynurenine (OH-KYN) with the same proficiency. Pre-steady-state kinetic analysis reveals that while the evolution of HsKYNase_66 is accompanied by a change in the rate-determining step of the reactions, HsKYNase_93D9 retains the same catalytic mechanism as HsKYNase. HDX-MS shows that the conformational dynamics of the two enzymes are markedly different and distinct from ortholog prokaryotic enzymes with high KYN activity. Our work provides a mechanistic framework for understanding the relationship between evolutionary mechanisms and phenotypic traits of evolved generalist and specialist enzyme species.

Suggested Citation

  • Christos S. Karamitros & Kyle Murray & Yoichi Kumada & Kenneth A. Johnson & Sheena D’Arcy & George Georgiou, 2024. "Mechanistic conformational and substrate selectivity profiles emerging in the evolution of enzymes via parallel trajectories," Nature Communications, Nature, vol. 15(1), pages 1-17, December.
  • Handle: RePEc:nat:natcom:v:15:y:2024:i:1:d:10.1038_s41467-024-51133-y
    DOI: 10.1038/s41467-024-51133-y
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    References listed on IDEAS

    as
    1. Dave W. Anderson & Florian Baier & Gloria Yang & Nobuhiko Tokuriki, 2021. "The adaptive landscape of a metallo-enzyme is shaped by environment-dependent epistasis," Nature Communications, Nature, vol. 12(1), pages 1-11, December.
    2. Renee Otten & Lin Liu & Lillian R. Kenner & Michael W. Clarkson & David Mavor & Dan S. Tawfik & Dorothee Kern & James S. Fraser, 2018. "Rescue of conformational dynamics in enzyme catalysis by directed evolution," Nature Communications, Nature, vol. 9(1), pages 1-11, December.
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