title: Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction.
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.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, 201 Gilman Hall, Berkeley, CA 94720-1462 (USA).|Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 5885 Hollis Street, Emeryville, CA 94608 (USA).
authors.forename: Sean
authors.initials: S
authors.lastname: Poust
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, 201 Gilman Hall, Berkeley, CA 94720-1462 (USA).|Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 5885 Hollis Street, Emeryville, CA 94608 (USA).
authors.forename: James
authors.initials: J
authors.lastname: Piety
authors.affiliation: Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm (Germany).
authors.forename: Arren
authors.initials: A
authors.lastname: Bar-Even
authors.affiliation: Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195 (USA).
authors.forename: Catherine
authors.initials: C
authors.lastname: Louw
authors.affiliation: Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195 (USA).
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of California, 201 Gilman Hall, Berkeley, CA 94720-1462 (USA).|Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, 5885 Hollis Street, Emeryville, CA 94608 (USA).
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
authors.affiliation: Departments of Chemistry and Biochemistry and Molecular Medicine, Genome Center, University of California, 451 Health Sciences Drive, Davis, CA 95616 (USA).|Department of Biochemistry, University of Washington, Box 357350, Seattle, WA 98195 (USA).
authors.forename: Justin B
authors.initials: JB
authors.lastname: Siegel
keywords: aldolases; carbon-carbon coupling; enzyme catalysis; molecular evolution; protein engineering