title: Robust design and optimization of retroaldol enzymes.
abstract: 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.
authors.affiliation: Department of Biochemistry, University of Washington and HHMI, Seattle, Washington 98195, USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Althoff
authors.forename: Ling
authors.initials: L
authors.lastname: Wang
authors.forename: Lin
authors.initials: L
authors.lastname: Jiang
authors.forename: Lars
authors.initials: L
authors.lastname: Giger
authors.forename: Jonathan K
authors.initials: JK
authors.lastname: Lassila
authors.forename: Zhizhi
authors.initials: Z
authors.lastname: Wang
authors.forename: Matthew
authors.initials: M
authors.lastname: Smith
authors.forename: Sanjay
authors.initials: S
authors.lastname: Hari
authors.forename: Peter
authors.initials: P
authors.lastname: Kast
authors.forename: Daniel
authors.initials: D
authors.lastname: Herschlag
authors.forename: Donald
authors.initials: D
authors.lastname: Hilvert
authors.forename: David
authors.initials: D
authors.lastname: Baker
mesh_terms: D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D019020:Directed Molecular Evolution / Q000379:methods; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D000446:Aldehyde-Lyases