De Novo Computational Design of Retro-Aldol Enzymes
Lin Jiang,1,2*
Eric A. Althoff,1*
Fernando R. Clemente,4
Lindsey Doyle,5
Daniela Röthlisberger,1
Alexandre Zanghellini,1,2
Jasmine L. Gallaher,1
Jamie L. Betker,1
Fujie Tanaka,6
Carlos F. Barbas, III,6
Donald Hilvert,7
Kendall N. Houk,4
Barry L. Stoddard,5
David Baker1,2,3
The creation of enzymes capable of catalyzing any desired chemical reaction is a grand challenge for computational protein design. Using new algorithms that rely on hashing techniques to construct active sites for multistep reactions, we designed retro-aldolases that use four different catalytic motifs to catalyze the breaking of a carbon-carbon bond in a nonnatural substrate. Of the 72 designs that were experimentally characterized, 32, spanning a range of protein folds, had detectable retro-aldolase activity. Designs that used an explicit water molecule to mediate proton shuffling were significantly more successful, with rate accelerations of up to four orders of magnitude and multiple turnovers, than those involving charged side-chain networks. The atomic accuracy of the design process was confirmed by the x-ray crystal structure of active designs embedded in two protein scaffolds, both of which were nearly superimposable on the design model.
1 Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
2 Biomolecular Structure and Design Program, University of Washington, Seattle, WA 98195, USA.
3 Howard Hughes Medical Institute (HHMI), University of Washington, Seattle, WA 98195, USA.
4 Department of Chemistry and Biochemistry, University of California, Los Angeles, CA 90095, USA.
5 Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
6 The Skaggs Institute for Chemical Biology and the Departments of Chemistry and Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
7 Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland.
* These authors contributed equally to this work.
To whom correspondence should be addressed. E-mail: dabaker{at}u.washington.edu
