Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction

Sean Poust, James Piety, Arren Bar-Even, Catherine Louw, David Baker, Jay D. Keasling, Justin Siegel

Research output: Contribution to journalArticle

15 Citations (Scopus)

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. To better understand the mechanism of the recently engineered formolase, mass spectrometry was used to characterize the enzyme's product profile, and showed that the partitioning between the two-carbon and the three-carbon products is controlled by formaldehyde concentration. Kinetic studies showed that the computationally designed residues were synergistic in their contributions towards enhancing activity.

Original languageEnglish (US)
Pages (from-to)1950-1954
Number of pages5
JournalChemBioChem
Volume16
Issue number13
DOIs
StatePublished - Sep 1 2015

Fingerprint

Carbon
Enzymes
Formaldehyde
Kinetics
Mass spectrometry
Mass Spectrometry
Dihydroxyacetone
Thermodynamics
Engineers
Mutation
formose sugars
Proteins
glycolaldehyde

Keywords

  • aldolases
  • carbon-carbon coupling
  • enzyme catalysis
  • molecular evolution
  • protein engineering

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology

Cite this

Poust, S., Piety, J., Bar-Even, A., Louw, C., Baker, D., Keasling, J. D., & Siegel, J. (2015). Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction. ChemBioChem, 16(13), 1950-1954. https://doi.org/10.1002/cbic.201500228

Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction. / Poust, Sean; Piety, James; Bar-Even, Arren; Louw, Catherine; Baker, David; Keasling, Jay D.; Siegel, Justin.

In: ChemBioChem, Vol. 16, No. 13, 01.09.2015, p. 1950-1954.

Research output: Contribution to journalArticle

Poust, S, Piety, J, Bar-Even, A, Louw, C, Baker, D, Keasling, JD & Siegel, J 2015, 'Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction', ChemBioChem, vol. 16, no. 13, pp. 1950-1954. https://doi.org/10.1002/cbic.201500228
Poust S, Piety J, Bar-Even A, Louw C, Baker D, Keasling JD et al. Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction. ChemBioChem. 2015 Sep 1;16(13):1950-1954. https://doi.org/10.1002/cbic.201500228
Poust, Sean ; Piety, James ; Bar-Even, Arren ; Louw, Catherine ; Baker, David ; Keasling, Jay D. ; Siegel, Justin. / Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction. In: ChemBioChem. 2015 ; Vol. 16, No. 13. pp. 1950-1954.
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