title: De novo computational design of retro-aldol enzymes.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.
authors.forename: Lin
authors.initials: L
authors.lastname: Jiang
authors.forename: Eric A
authors.initials: EA
authors.lastname: Althoff
authors.forename: Fernando R
authors.initials: FR
authors.lastname: Clemente
authors.forename: Lindsey
authors.initials: L
authors.lastname: Doyle
authors.forename: Daniela
authors.initials: D
authors.lastname: Röthlisberger
authors.forename: Alexandre
authors.initials: A
authors.lastname: Zanghellini
authors.forename: Jasmine L
authors.initials: JL
authors.lastname: Gallaher
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Betker
authors.forename: Fujie
authors.initials: F
authors.lastname: Tanaka
authors.forename: Carlos F
authors.initials: CF
authors.lastname: Barbas
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
authors.forename: Kendall N
authors.initials: KN
authors.lastname: Houk
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
authors.forename: David
authors.initials: D
authors.lastname: Baker
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000378:metabolism; D000465:Algorithms; D001665:Binding Sites; D002384:Catalysis; D020134:Catalytic Domain; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D000446:Aldehyde-Lyases