ArticleProceedings of the National Academy of Sciences of the United States of America2012
Bridging the gaps in design methodologies by evolutionary optimization of the stability and proficiency of designed Kemp eliminase KE59.
Article in Proceedings of the National Academy of Sciences of the United States of America, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 84 papers.
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84 citing papers in PubMed, 238 citations in OpenAlex.
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- Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release.Nature communications · 2025Article
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- Enriching productive mutational paths accelerates enzyme evolution.Nature chemical biology · 2024Article
- Substrate Positioning Dynamics Involves a Non-Electrostatic Component to Mediate Catalysis.The journal of physical chemistry letters · 2023Article
- Mutexa: A Computational Ecosystem for Intelligent Protein Engineering.Journal of chemical theory and computation · 2023Review
- Design of efficient artificial enzymes using crystallographically-enhanced conformational sampling.bioRxiv : the preprint server for biology · 2023Article
- Fishing for Catalysis: Experimental Approaches to Narrowing Search Space in Directed Evolution of Enzymes.JACS Au · 2023Review
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- Data-driven enzyme engineering to identify function-enhancing enzymes.Protein engineering, design & selection : PEDS · 2023Review
- Transient binding sites at the surface of haloalkane dehalogenase LinB as locations for fine-tuning enzymatic activity.PloS one · 2023Article
- Convergence in determining enzyme functional descriptors across Kemp eliminase variants.Electronic structure (Bristol, England) · 2022Article
- Active site center redesign increases protein stability preserving catalysis in thioredoxin.Protein science : a publication of the Protein Society · 2022Article
- Assessing and enhancing foldability in designed proteins.Protein science : a publication of the Protein Society · 2022Article
- Natural Evolution Provides Strong Hints about Laboratory Evolution of Designer Enzymes.Proceedings of the National Academy of Sciences of the United States of America · 2022Article
- What Have We Learned from Design of Function in Large Proteins?Biodesign research · 2022Review
24 more citing papers are in PubMed but not listed here.
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Authors and funding
8 authors at 3 institutions in 2 countries.
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No grant is acknowledged in the PubMed record.
Abstract
Computational design is a test of our understanding of enzyme catalysis and a means of engineering novel, tailor-made enzymes. While the de novo computational design of catalytically efficient enzymes remains a challenge, designed enzymes may comprise unique starting points for further optimization by directed evolution. Directed evolution of two computationally designed Kemp eliminases, KE07 and KE70, led to low to moderately efficient enzymes (k(cat)/K(m) values of ≤ 5 10(4) M(-1)s(-1)). Here we describe the optimization of a third design, KE59. Although KE59 was the most catalytically efficient Kemp eliminase from this design series (by k(cat)/K(m), and by catalyzing the elimination of nonactivated benzisoxazoles), its impaired stability prevented its evolutionary optimization. To boost KE59's evolvability, stabilizing consensus mutations were included in the libraries throughout the directed evolution process. The libraries were also screened with less activated substrates. Sixteen rounds of mutation and selection led to > 2,000-fold increase in catalytic efficiency, mainly via higher k(cat) values. The best KE59 variants exhibited k(cat)/K(m) values up to 0.6 10(6) M(-1)s(-1), and k(cat)/k(uncat) values of ≤ 10(7) almost regardless of substrate reactivity. Biochemical, structural, and molecular dynamics (MD) simulation studies provided insights regarding the optimization of KE59. Overall, the directed evolution of three different designed Kemp eliminases, KE07, KE70, and KE59, demonstrates that computational designs are highly evolvable and can be optimized to high catalytic efficiencies.
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