ArticleJournal of molecular biology2011
Optimization of the in-silico-designed kemp eliminase KE70 by computational design and directed evolution.
Article in Journal of molecular biology, 2011. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 60 papers.
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Who cites it
60 citing papers in PubMed, 180 citations in OpenAlex.
- Predicting Activation Barriers With Ring and Bond Critical Points in the Kemp Elimination Reaction.Chemphyschem : a European journal of chemical physics and physical chemistry · 2026Article
- Emergence of specific binding and catalysis from a designed generalist binding protein.Nature chemistry · 2026Article
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- Protein engineering: status report.Protein engineering, design & selection : PEDS · 2026Review
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- Distal mutations enhance catalysis in designed enzymes by facilitating substrate binding and product release.Nature communications · 2025Article
- Fluorogenic, Subsingle-Turnover Monitoring of Enzymatic Reactions Involving NAD(P)H Provides a Generalized Platform for Directed Ultrahigh-Throughput Evolution of Biocatalysts in Microdroplets.Journal of the American Chemical Society · 2025Article
- Perspectives on Computational Enzyme Modeling: From Mechanisms to Design and Drug Development.ACS omega · 2024Review
- Theoretical Improvements in Enzyme Efficiency Associated with Noisy Rate Constants and Increased Dissipation.Entropy (Basel, Switzerland) · 2024Article
- 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
- AsiteDesign: a Semirational Algorithm for an Automated Enzyme Design.The journal of physical chemistry. B · 2023Article
- 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
- Making Enzymes Suitable for Organic Chemistry by Rational Protein Design.Chembiochem : a European journal of chemical biology · 2022Review
- Evolution of Enzyme Function and the Development of Catalytic Efficiency: Triosephosphate Isomerase, Jeremy R. Knowles, and W. John Albery.Biochemistry · 2021Article
- Current state of and need for enzyme engineering of 2-deoxy-D-ribose 5-phosphate aldolases and its impact.Applied microbiology and biotechnology · 2021Review
- Development of a yeast cell surface display method using the SpyTag/SpyCatcher system.Scientific reports · 2021Article
- Catalytic Principles from Natural Enzymes and Translational Design Strategies for Synthetic Catalysts.ACS central science · 2021Review
- How directed evolution reshapes the energy landscape in an enzyme to boost catalysis.Science (New York, N.Y.) · 2020Article
- Tight and specific lanthanide binding in a de novo TIM barrel with a large internal cavity designed by symmetric domain fusion.Proceedings of the National Academy of Sciences of the United States of America · 2020Article
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Authors and funding
10 authors at 3 institutions in 2 countries.
Funding
Abstract
Although de novo computational enzyme design has been shown to be feasible, the field is still in its infancy: the kinetic parameters of designed enzymes are still orders of magnitude lower than those of naturally occurring ones. Nonetheless, designed enzymes can be improved by directed evolution, as recently exemplified for the designed Kemp eliminase KE07. Random mutagenesis and screening resulted in variants with >200-fold higher catalytic efficiency and provided insights about features missing in the designed enzyme. Here we describe the optimization of KE70, another designed Kemp eliminase. Amino acid substitutions predicted to improve catalysis in design calculations involving extensive backbone sampling were individually tested. Those proven beneficial were combinatorially incorporated into the originally designed KE70 along with random mutations, and the resulting libraries were screened for improved eliminase activity. Nine rounds of mutation and selection resulted in >400-fold improvement in the catalytic efficiency of the original KE70 design, reflected in both higher k(cat) values and lower K(m) values, with the best variants exhibiting k(cat)/K(m) values of >5×10(4) s(-)(1) M(-1). The optimized KE70 variants were characterized structurally and biochemically, providing insights into the origins of the improvements in catalysis. Three primary contributions were identified: first, the reshaping of the active-site cavity to achieve tighter substrate binding; second, the fine-tuning of electrostatics around the catalytic His-Asp dyad; and, third, the stabilization of the active-site dyad in a conformation optimal for catalysis.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.