Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.
By combining targeted mutagenesis, computational refinement, and directed evolution, a modestly active, computationally designed Diels-Alderase was converted into the most proficient biocatalyst for [4+2] cycloadditions known. The high stereoselectivity and minimal product inhibition of the evolved enzyme enabled preparative scale synthesis of a single product diastereomer. X-ray crystallography of the enzyme-product complex shows that the molecular changes introduced over the course of optimization, including addition of a lid structure, gradually reshaped the pocket for more effective substrate preorganization and transition state stabilization. The good overall agreement between the experimental structure and the original design model with respect to the orientations of both the bound product and the catalytic side chains contrasts with other computationally designed enzymes. Because design accuracy appears to correlate with scaffold rigidity, improved control over backbone conformation will likely be the key to future efforts to design more efficient enzymes for diverse chemical reactions.
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
Nathalie
N
Preiswerk
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
Tobias
T
Beck
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
Jessica D
JD
Schulz
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
Peter
P
Milovník
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland;
Clemens
C
Mayer
Department of Biochemistry, University of Washington, Seattle, WA 98195; andDepartments of Biochemistry and Molecular Medicine andChemistry, andGenome Center, University of California, Davis, CA 95616.
Justin B
JB
Siegel
Department of Biochemistry, University of Washington, Seattle, WA 98195; and.
David
D
Baker
Laboratory of Organic Chemistry, Eidgenössische Technische Hochschule Zürich, 8093 Zurich, Switzerland; hilvert@org.chem.ethz.ch.
Donald
D
Hilvert
D000178:Acrylamides / Q000737:chemistry; D002070:Butadienes / Q000737:chemistry; D002384:Catalysis; D003460:Crystallization; D018360:Crystallography, X-Ray; D061565:Cycloaddition Reaction / Q000379:methods*; D004789:Enzyme Activation; D004798:Enzymes / Q000138:chemical synthesis* / Q000737:chemistry*; D019418:Evolution, Chemical; D007700:Kinetics; D008956:Models, Chemical; D013379:Substrate Specificity
C551201:4-carboxybenzyl trans-1,3-butadiene-1-carbamate; D000178:Acrylamides; D002070:Butadienes; D004798:Enzymes; C099046:N,N-dimethylacrylamide
Diels–Alder reaction; biocatalysis; computational enzyme design; enzyme mechanism; laboratory evolution