title: The use of ene adducts to study and engineer enoyl-thioester reductases.
abstract: An improved understanding of enzymes' catalytic proficiency and stereoselectivity would further enable applications in chemistry, biocatalysis and industrial biotechnology. We use a chemical probe to dissect individual catalytic steps of enoyl-thioester reductases (Etrs), validating an active site tyrosine as the cryptic proton donor and explaining how it had eluded definitive identification. This information enabled the rational redesign of Etr, yielding mutants that create products with inverted stereochemistry at wild type-like turnover frequency.
authors: Institute of Microbiology, ETH (Swiss Federal Institute of Technology) Zurich, Zurich, Switzerland.
authors: Raoul G
authors: RG
authors: Rosenthal
authors: Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
authors: Bastian
authors: B
authors: Vögeli
authors: Institute of Molecular Biology and Biophysics, ETH Zurich, Zurich, Switzerland.
authors: Nick
authors: N
authors: Quade
authors: Paul Scherrer Institute, Villigen, Switzerland.
authors: Guido
authors: G
authors: Capitani
authors: Institute of Microbiology, ETH (Swiss Federal Institute of Technology) Zurich, Zurich, Switzerland.
authors: Patrick
authors: P
authors: Kiefer
authors: Institute of Microbiology, ETH (Swiss Federal Institute of Technology) Zurich, Zurich, Switzerland.
authors: Julia A
authors: JA
authors: Vorholt
authors: Laboratory of Organic Chemistry, ETH Zurich, Zurich, Switzerland. .
authors: Marc-Olivier
authors: MO
authors: Ebert
authors: 1] Institute of Microbiology, ETH (Swiss Federal Institute of Technology) Zurich, Zurich, Switzerland. [2] Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.
authors: Tobias J
authors: TJ
authors: Erb
mesh_terms: D001665:Binding Sites; D001709:Biotechnology / Q000379:methods*; D002384:Catalysis; D008958:Models, Molecular; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000737:chemistry* / Q000235:genetics*; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011522:Protons; D013237:Stereoisomerism; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
chemical_list: D011522:Protons; D014443:Tyrosine; D044925:Oxidoreductases Acting on CH-CH Group Donors; C434934:trans-2-enoyl-CoA reductase (NADPH)