title: A redox-mediated Kemp eliminase.
abstract: The acid/base-catalysed Kemp elimination of 5-nitro-benzisoxazole forming 2-cyano-4-nitrophenol has long served as a design platform of enzymes with non-natural reactions, providing new mechanistic insights in protein science. Here we describe an alternative concept based on redox catalysis by P450-BM3, leading to the same Kemp product via a fundamentally different mechanism. QM/MM computations show that it involves coordination of the substrate's N-atom to haem-Fe(II) with electron transfer and concomitant N-O heterolysis liberating an intermediate having a nitrogen radical moiety Fe(III)-N· and a phenoxyl anion. Product formation occurs by bond rotation and H-transfer. Two rationally chosen point mutations cause a notable increase in activity. The results shed light on the prevailing mechanistic uncertainties in human P450-catalysed metabolism of the immunomodulatory drug leflunomide, which likewise undergoes redox-mediated Kemp elimination by P450-BM3. Other isoxazole-based pharmaceuticals are probably also metabolized by a redox mechanism. Our work provides a basis for designing future artificial enzymes.
authors.affiliation: Department of Biocatalysis, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, Mülheim an der Ruhr 45470, Germany.|Department of Chemistry, Philipps-Universität Marburg, Marburg 35032, Germany.
authors.forename: Aitao
authors.initials: A
authors.lastname: Li
authors.affiliation: Institute of Chemistry and the Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
authors.forename: Binju
authors.initials: B
authors.lastname: Wang
authors.affiliation: Department of Biocatalysis, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, Mülheim an der Ruhr 45470, Germany.|Department of Chemistry, Philipps-Universität Marburg, Marburg 35032, Germany.
authors.forename: Adriana
authors.initials: A
authors.lastname: Ilie
authors.affiliation: Institute of Chemistry and the Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
authors.forename: Kshatresh D
authors.identifier: 0000-0001-8865-7602
authors.initials: KD
authors.lastname: Dubey
authors.affiliation: LOEWE Center for Synthetic Microbiology (SYNMIKRO) and Department of Chemistry, Philipps-Universität Marburg, Marburg 35032, Germany.
authors.forename: Gert
authors.initials: G
authors.lastname: Bange
authors.affiliation: Department of Chemistry, Syracuse University, 111 College Place, Syracuse, New York 13244, USA.
authors.forename: Ivan V
authors.initials: IV
authors.lastname: Korendovych
authors.affiliation: Institute of Chemistry and the Lise Meitner-Minerva Center for Computational Quantum Chemistry, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel.
authors.forename: Sason
authors.initials: S
authors.lastname: Shaik
authors.affiliation: Department of Biocatalysis, Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, Mülheim an der Ruhr 45470, Germany.|Department of Chemistry, Philipps-Universität Marburg, Marburg 35032, Germany.
authors.forename: Manfred T
authors.initials: MT
authors.lastname: Reetz
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology*; D055162:Biocatalysis; D002384:Catalysis; D020134:Catalytic Domain; D055598:Chemical Phenomena; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D006801:Humans; D007555:Isoxazoles / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D000077339:Leflunomide; D056004:Molecular Dynamics Simulation; D010084:Oxidation-Reduction; D015202:Protein Engineering; D011789:Quantum Theory; D013379:Substrate Specificity
chemical_list: D007555:Isoxazoles; D003577:Cytochrome P-450 Enzyme System; D000077339:Leflunomide