AUTHOR=Sosa Yvett , Kapur Bhav , Hurtak Jessica , Kingsley Laura J. , Wu Hao , Gruber Stefanie , Nar Herbert , Khattabi Saad , Moral Jesus Seco , Lucas Maria Fátima , Martin Caterina , Lončar Nikola , Buono Frederic , Pefaur Noah , Nixon Andrew E. , Song Jinhua J.
TITLE=In silico enzyme screening identifies an SDR ketoreductase from Thermus caliditerrae as an attractive biocatalyst and promising candidate for protein engineering
JOURNAL=Frontiers in Chemical Biology
VOLUME=3
YEAR=2024
URL=https://www.frontiersin.org/journals/chemical-biology/articles/10.3389/fchbi.2024.1425501
DOI=10.3389/fchbi.2024.1425501
ISSN=2813-530X
ABSTRACT=Introduction: Biocatalysis, particularly through engineered enzymes, presents a cost-effective, efficient, and eco-friendly 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.
Methods: 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.
Results: 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.
Conclusion: The enantioselectivity combined with the thermostability of Tcalid SDR makes this enzyme an attractive engineering starting point for biocatalysis applications.