Robust design and optimization of retroaldol enzymes.
|Title||Robust design and optimization of retroaldol enzymes.|
|Publication Type||Journal Article|
|Year of Publication||2012|
|Authors||Althoff, E. A., Wang L., Jiang L., Giger L., Lassila J. K., Wang Z., Smith M., Hari S., Kast P., Herschlag D., Hilvert D., & Baker D.|
|Journal||Protein science : a publication of the Protein Society|
|Date Published||2012 May|
|Keywords||Aldehyde-Lyases, Catalytic Domain, Directed Molecular Evolution, Models, Molecular, Mutagenesis, Site-Directed, Primary Publication, Protein Engineering, Recombinant Proteins|
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.
|Alternate Journal||Protein Sci.|