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Mechanistic Analysis of an Engineered Enzyme that Catalyzes the Formose Reaction
Author(s) -
Poust Sean,
Piety James,
BarEven Arren,
Louw Catherine,
Baker David,
Keasling Jay D.,
Siegel Justin B.
Publication year - 2015
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201500228
Subject(s) - dihydroxyacetone , glycolaldehyde , chemistry , biocatalysis , enzyme , yield (engineering) , formaldehyde , computational chemistry , combinatorial chemistry , biochemistry , reaction mechanism , catalysis , materials science , metallurgy , glycerol
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.