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ORIGINAL RESEARCH article

Front. Chem. Biol.
Sec. Theoretical Modeling, Structure Prediction & Design
Volume 3 - 2024 | doi: 10.3389/fchbi.2024.1425501
This article is part of the Research Topic Structure-function relationship of enzymes through a chemical lens View all 5 articles

In silico enzyme screening identifies an SDR ketoreductase from Thermus caliditerrae as an attractive biocatalyst and promising candidate for protein engineering

Provisionally accepted
Yvett Sosa Yvett Sosa 1*Bhav Kapur Bhav Kapur 2Jessica Hurtak Jessica Hurtak 3Laura Kingsley Laura Kingsley 4*Hao Wu Hao Wu 3*Stefanie Gruber Stefanie Gruber 2*Herbert Nar Herbert Nar 2Saad Khattabi Saad Khattabi 3*Jesus Seco Moral Jesus Seco Moral 5Maria Fatima Lucas Maria Fatima Lucas 5*Caterina Martin Caterina Martin 6Nikola Lončar Nikola Lončar 6*frederic buono frederic buono 3*Noah Pefaur Noah Pefaur 4Andrew E. Nixon Andrew E. Nixon 4*Jeff Song Jeff Song 3*
  • 1 Chemical Development, Biotherapeutics, Boehringer Ingelheim (United States), Ridgefield, United States
  • 2 Structural Biology, Boehringer-Ingelheim, Biberach, Germany
  • 3 Chemical Development, Boehringer Ingelheim (United States), Ridgefield, United States
  • 4 Biotherapeutics, Boehringer Ingelheim (United States), Ridgefield, United States
  • 5 zymvol, Barcelona, Spain
  • 6 Gecco-Biotech, Groningen, Netherlands

The final, formatted version of the article will be published soon.

    Biocatalysis, particularly through engineered enzymes, presents a cost-effective, efficient, and ecofriendly approach to compound synthesis. We sought to identify ketoreductases capable of synthesizing optically pure alcohols or ketones, essential chiral building blocks for active pharmaceutical ingredients. Using BioMatchMaker®, an in silico high-throughput platform that allows the identification of wild-type enzyme sequences for a desired chemical transformation, we identified a bacterial SDR ketoreductase from Thermus caliditerrae, Tcalid SDR, that demonstrates favorable reaction efficiency and desired enantiomeric excess. Here we present two crystal structures of the Tcalid SDR in an apo-form at 1.9 Å and NADP-complexed form at 1.7 Å resolution (9FE6 and 9FEB, respectively). This enzyme forms a homotetramer with each subunit containing an N-terminal Rossmann-fold domain. We use computational analysis combined with site-directed mutagenesis and enzymatic characterization to define the substrate-binding pocket. Furthermore, the enzyme retained favorable reactivity and selectivity after incubation at elevated temperature. The enantioselectivity combined with the thermostability of Tcalid SDR makes this enzyme an attractive engineering starting point for biocatalysis applications.

    Keywords: ketoreductase, kinetic resolution, In silico screening, asymmetric synthesis, BioMatchMaker, Biocatalysis, Thermus caliditerrae

    Received: 29 Apr 2024; Accepted: 12 Jun 2024.

    Copyright: © 2024 Sosa, Kapur, Hurtak, Kingsley, Wu, Gruber, Nar, Khattabi, Seco Moral, Lucas, Martin, Lončar, buono, Pefaur, Nixon and Song. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

    * Correspondence:
    Yvett Sosa, Chemical Development, Biotherapeutics, Boehringer Ingelheim (United States), Ridgefield, United States
    Laura Kingsley, Biotherapeutics, Boehringer Ingelheim (United States), Ridgefield, United States
    Hao Wu, Chemical Development, Boehringer Ingelheim (United States), Ridgefield, United States
    Stefanie Gruber, Structural Biology, Boehringer-Ingelheim, Biberach, Germany
    Saad Khattabi, Chemical Development, Boehringer Ingelheim (United States), Ridgefield, United States
    Maria Fatima Lucas, zymvol, Barcelona, Spain
    Nikola Lončar, Gecco-Biotech, Groningen, Netherlands
    frederic buono, Chemical Development, Boehringer Ingelheim (United States), Ridgefield, United States
    Andrew E. Nixon, Biotherapeutics, Boehringer Ingelheim (United States), Ridgefield, United States
    Jeff Song, Chemical Development, Boehringer Ingelheim (United States), Ridgefield, United States

    Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.