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Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction |
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| Authors: | Sean Poust James Piety Dr. Arren Bar‐Even Dr. Catherine Louw Prof. Dr. David Baker Prof. Dr. Jay D. Keasling Prof. Dr. Justin B. Siegel |
| Affiliation: | 1. Department of Chemical and Biomolecular Engineering, University of California, 201 Gilman Hall, Berkeley, CA 94720‐1462 (USA);2. Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 5885 Hollis Street, Emeryville, CA 94608 (USA);3. Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam‐Golm (Germany);4. Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195 (USA);5. Departments of Chemistry and Biochemistry and Molecular Medicine, Genome Center, University of California, 451 Health Sciences Drive, Davis, CA 95616 (USA) |
| Abstract: | An enzyme that catalyzes the formose reaction, termed “formolase”, was recently engineered through a combination of computational protein design and directed evolution. We have investigated the kinetic role of the computationally designed residues and further characterized the enzyme's product profile. Kinetic studies illustrated that the computationally designed mutations were synergistic in their contributions towards enhancing activity. Mass spectrometry revealed that the engineered enzyme produces two products of the formose reaction—dihydroxyacetone and glycolaldehyde—with the product profile dependent on the formaldehyde concentration. We further explored the effects of this product profile on the thermodynamics and yield of the overall carbon assimilation from the formolase pathway to help guide future efforts to engineer this pathway. |
| Keywords: | aldolases carbon– carbon coupling enzyme catalysis molecular evolution protein engineering |