title: Kemp elimination catalysts by computational enzyme design.
abstract: The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. Here we describe the computational design of eight enzymes that use two different catalytic motifs to catalyse the Kemp elimination-a model reaction for proton transfer from carbon-with measured rate enhancements of up to 10(5) and multiple turnovers. Mutational analysis confirms that catalysis depends on the computationally designed active sites, and a high-resolution crystal structure suggests that the designs have close to atomic accuracy. Application of in vitro evolution to enhance the computational designs produced a >200-fold increase in k(cat)/K(m) (k(cat)/K(m) of 2,600 M(-1)s(-1) and k(cat)/k(uncat) of >10(6)). These results demonstrate the power of combining computational protein design with directed evolution for creating new enzymes, and we anticipate the creation of a wide range of useful new catalysts in the future.
authors: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.
authors: Daniela
authors: D
authors: Röthlisberger
authors: Olga
authors: O
authors: Khersonsky
authors: Andrew M
authors: AM
authors: Wollacott
authors: Lin
authors: L
authors: Jiang
authors: Jason
authors: J
authors: DeChancie
authors: Jamie
authors: J
authors: Betker
authors: Jasmine L
authors: JL
authors: Gallaher
authors: Eric A
authors: EA
authors: Althoff
authors: Alexandre
authors: A
authors: Zanghellini
authors: Orly
authors: O
authors: Dym
authors: Shira
authors: S
authors: Albeck
authors: Kendall N
authors: KN
authors: Houk
authors: Dan S
authors: DS
authors: Tawfik
authors: David
authors: D
authors: Baker
mesh_terms: D000465:Algorithms; D020816:Amino Acid Motifs; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D019295:Computational Biology; D003198:Computer Simulation; D018360:Crystallography, X-Ray; D019020:Directed Molecular Evolution / Q000379:methods*; D015195:Drug Design; D004353:Drug Evaluation, Preclinical; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D015202:Protein Engineering / Q000379:methods*; D011789:Quantum Theory; D012680:Sensitivity and Specificity
chemical_list: D004798:Enzymes