title: Characterization of a new Baeyer-Villiger monooxygenase and conversion to a solely N-or S-oxidizing enzyme by a single R292 mutation.
abstract: Ar-BVMO is a recently discovered Baeyer-Villiger monooxygenase from the genome of Acinetobacter radioresistens S13 closely related to medically relevant ethionamide monooxygenase EtaA (prodrug activator) and capable of inactivating the imipenem antibiotic.
  
  
  
  The co-substrate preference as well as steady-state and rapid kinetics studies of the recombinant purified protein were carried out using stopped-flow spectroscopy under anaerobic and aerobic conditions. Kd values were measured by isothermal calorimetry. Enzymatic activity was determined by measuring the amount of product formed using high pressure liquid chromatography or gas chromatography. Site-directed mutagenesis experiments were performed to decipher the role of the active site arginine-292.
  
  
  
  Ar-BVMO was found to oxidize ethionamide as well as linear ketones. Mechanistic studies on the wild type enzyme using stopped-flow spectroscopy allowed for the detection of the characteristic oxygenating C4a-(hydro)peroxyflavin intermediate, which decayed rapidly in the presence of the substrate. Replacement of arginine 292 in Ar-BVMO by glycine or alanine resulted in greatly reduced or no Baeyer-Villiger activity, respectively, demonstrating the crucial role of this residue in catalysis of ketone substrates. However, both the R292A and R292G mutants are capable of carrying out N- and S-oxidation reactions.
  
  
  
  Substrate profiling of Ar-BVMO confirms its close relationship to EtaA; ethionamide is one of its substrates. The active site Arginine 292 is required for its Baeyer-Villiger activity but not for heteroatom oxidation.
  
  
  
  A single mutation converts Ar-BVMO to a unique S- or N-monooxygenase, a useful biocatalyst for the production of oxidized metabolites of human drug metabolizing enzymes.
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Catucci
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Ivan
authors.initials: I
authors.lastname: Zgrablic
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Lanciani
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Valetti
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Minerdi
authors.affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109-0600, USA.
authors.forename: David P
authors.initials: DP
authors.lastname: Ballou
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Gilardi
authors.affiliation: Department of Life Sciences and Systems Biology, University of Torino, 10123 Turin, Italy. Electronic address: sheila.sadeghi@unito.it.
authors.forename: Sheila J
authors.initials: SJ
authors.lastname: Sadeghi
mesh_terms: D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005000:Ethionamide / Q000737:chemistry* / Q000378:metabolism; D005415:Flavins / Q000737:chemistry* / Q000378:metabolism; D015870:Gene Expression; D005998:Glycine / Q000737:chemistry / Q000378:metabolism; D007659:Ketones / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; C531259:C4a-hydroperoxyflavin; D005415:Flavins; D007659:Ketones; D011994:Recombinant Proteins; D001120:Arginine; D006899:Mixed Function Oxygenases; D005000:Ethionamide; D000409:Alanine; D005998:Glycine
keywords: BVMO; Drug metabolites; Ethionamide; Flavoprotein; Isothermal calorimetry; Stopped-flow