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Robust design and optimization of retroaldol enzymes
Author(s) -
Althoff Eric A.,
Wang Ling,
Jiang Lin,
Giger Lars,
Lassila Jonathan K.,
Wang Zhizhi,
Smith Matthew,
Hari Sanjay,
Kast Peter,
Herschlag Daniel,
Hilvert Donald,
Baker David
Publication year - 2012
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.2059
Subject(s) - lysine , catalysis , chemistry , enzyme , combinatorial chemistry , protein design , mutagenesis , protein engineering , rational design , borohydride , directed evolution , active site , enzyme catalysis , computer science , biochemistry , protein structure , mutant , nanotechnology , materials science , amino acid , gene
Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water‐mediated stabilization of the carbinolamine hydroxyl and β‐hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site‐directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride‐reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.