title: Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis.
abstract: BACKGROUND
  OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism.
  
  
  METHODS
  The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent.
  
  
  RESULTS
  Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates.
  
  
  CONCLUSIONS
  Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP.
  
  
  CONCLUSIONS
  Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C–C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy; Department of Biochemical Sciences, "Sapienza" University of Rome, P.le A. Moro 5, 00185 Rome, Italy.
authors.forename: Linda Celeste
authors.initials: LC
authors.lastname: Montemiglio
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy; Department of Biochemical Sciences, "Sapienza" University of Rome, P.le A. Moro 5, 00185 Rome, Italy.
authors.forename: Giacomo
authors.initials: G
authors.lastname: Parisi
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy; Department of Biochemical Sciences, "Sapienza" University of Rome, P.le A. Moro 5, 00185 Rome, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Scaglione
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy; Department of Biochemical Sciences, "Sapienza" University of Rome, P.le A. Moro 5, 00185 Rome, Italy.
authors.forename: Giuliano
authors.initials: G
authors.lastname: Sciara
authors.affiliation: CNR Institute of Molecular Biology and Pathology, P.le A. Moro 5, 00185 Rome, Italy. Electronic address: linda.savino@uniroma1.it.
authors.forename: Carmelinda
authors.initials: C
authors.lastname: Savino
authors.affiliation: Istituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Scienze Biochimiche "A. Rossi Fanelli", Sapienza Università di Roma, P.le A. Moro 5, 00185 Rome, Italy; Department of Biochemical Sciences, "Sapienza" University of Rome, P.le A. Moro 5, 00185 Rome, Italy; CNR Institute of Molecular Biology and Pathology, P.le A. Moro 5, 00185 Rome, Italy. Electronic address: beatrice.vallone@uniroma1.it.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Vallone
mesh_terms: D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D020134:Catalytic Domain; D003022:Clotrimazole / Q000737:chemistry*; D003461:Crystallography; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000502:physiology; D009827:Oleandomycin / Q000096:biosynthesis*; D010088:Oxidoreductases / Q000737:chemistry* / Q000502:physiology; D017433:Protein Structure, Secondary; D013303:Streptomyces antibioticus / Q000201:enzymology*; D013329:Structure-Activity Relationship
chemical_list: D000900:Anti-Bacterial Agents; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C023111:epoxidase; D003022:Clotrimazole; D009827:Oleandomycin
keywords: Clotrimazole and Ketoconazole; Crystal structure; Cyp107d1; Epoxigenases; Oleandomycin; P450