title: Identification of promiscuous ene-reductase activity by mining structural databases using active site constellations.
abstract: The exploitation of catalytic promiscuity and the application of de novo design have recently opened the access to novel, non-natural enzymatic activities. Here we describe a structural bioinformatic method for predicting catalytic activities of enzymes based on three-dimensional constellations of functional groups in active sites ('catalophores'). As a proof-of-concept we identify two enzymes with predicted promiscuous ene-reductase activity (reduction of activated C-C double bonds) and compare them with known ene-reductases, that is, members of the Old Yellow Enzyme family. Despite completely different amino acid sequences, overall structures and protein folds, high-resolution crystal structures reveal equivalent binding modes of typical Old Yellow Enzyme substrates and ligands. Biochemical and biocatalytic data show that the two enzymes indeed possess ene-reductase activity and reveal an inverted stereopreference compared with Old Yellow Enzymes for some substrates. This method could thus be a tool for the identification of viable starting points for the development and engineering of novel biocatalysts.
authors: 1] ACIB GmbH, Petersgasse 14, 8010 Graz, Austria [2].
authors: Georg
authors: G
authors: Steinkellner
authors: 1] ACIB GmbH, Petersgasse 14, 8010 Graz, Austria [2].
authors: Christian C
authors: CC
authors: Gruber
authors: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria.
authors: Tea
authors: T
authors: Pavkov-Keller
authors: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria.
authors: Alexandra
authors: A
authors: Binter
authors: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria.
authors: Kerstin
authors: K
authors: Steiner
authors: Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria.
authors: Christoph
authors: C
authors: Winkler
authors: ACIB GmbH, Petersgasse 14, 8010 Graz, Austria.
authors: Andrzej
authors: A
authors: Lyskowski
authors: 1] ACIB GmbH, Petersgasse 14, 8010 Graz, Austria [2] Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50, 8010 Graz, Austria.
authors: Orsolya
authors: O
authors: Schwamberger
authors: Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50, 8010 Graz, Austria.
authors: Monika
authors: M
authors: Oberer
authors: 1] ACIB GmbH, Petersgasse 14, 8010 Graz, Austria [2] Institute of Molecular Biotechnology, Graz University of Technology, Petersgasse 14, 8010 Graz, Austria.
authors: Helmut
authors: H
authors: Schwab
authors: 1] ACIB GmbH, Petersgasse 14, 8010 Graz, Austria [2] Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria.
authors: Kurt
authors: K
authors: Faber
authors: 1] ACIB GmbH, Petersgasse 14, 8010 Graz, Austria [2] Institute of Biochemistry, Graz University of Technology, Petersgasse 12, 8010 Graz, Austria.
authors: Peter
authors: P
authors: Macheroux
authors: 1] ACIB GmbH, Petersgasse 14, 8010 Graz, Austria [2] Institute of Molecular Biosciences, University of Graz, Humboldtstrasse 50, 8010 Graz, Austria.
authors: Karl
authors: K
authors: Gruber
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D030562:Databases, Protein; D007700:Kinetics; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D016963:Thermus thermophilus / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D010088:Oxidoreductases