Impact of scaffold rigidity on the design and evolution of an artificial Diels-Alderase.

ABSTRACT: 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 p

SUBMITTER: Preiswerk N 

PROVIDER: S-EPMC4050586 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature