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Complete switch of reaction specificity of an aldolase by directed evolution in vitro: Synthesis of generic aliphatic aldol products

AuthorsJunker, Sebastian; Roldán, Raquel; Joosten, Henk-Jan; Clapés Saborit, Pere ; Fessner, Wolf-Dieter
Fructose-6-phosphate aldolase
HPTLC screening
Protein Engineering
Issue Date8-Jun-2018
CitationAngewandte Chemie - International Edition
AbstractA structure‐guided engineering of fructose‐6‐phosphate aldolase was performed to expand its substrate promiscuity toward aliphatic nucleophiles, i.e., unsubstituted alkanones and alkanals. A "smart" combinatorial library was created targeting residues D6, T26 and N28 that form a binding pocket around the nucleophilic carbon atom. Double‐selectivity screening was executed by high‐performance TLC that allowed simultaneous determination of total activity as well as a preference for acetone versus propanal as competing nucleophiles. While any mutation of N28 resulted in inactivation of the enzyme, D6 turned out to be the key residue that enabled activity with non‐hydroxylated nucleophiles. Altogether 25 single‐ and double‐site variants (D6X and D6X/T26X) were discovered that show useful synthetic activity and a varying preference for ketone or aldehyde as the aldol nucleophiles. Remarkably, all of the novel variants had completely lost their native activity for cleavage of fructose 6‐phosphate.
Publisher version (URL)https://doi.org/10.1002/anie.201804831
Appears in Collections:(IQAC) Artículos
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