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The 1.92-Å Structure of Streptomyces coelicolor A3(2) CYP154C1

A NEW MONOOXYGENASE THAT FUNCTIONALIZES MACROLIDE RING SYSTEMS*
  • Larissa M. Podust
    Correspondence
    To whom correspondence should be addressed: Dept. of Biochemistry, Vanderbilt University, 23rd South at Pierce, Nashville, TN 37232-0146. Tel.: 615-343-4644; Fax: 615-322-4349
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Youngchang Kim
    Affiliations
    Argonne National Laboratory, Structural Biology Center, Argonne, Illinois 60439
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  • Miharu Arase
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Benjamin A. Neely
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Brian J. Beck
    Affiliations
    Department of Microbiology and BioTechnology Institute, University of Minnesota, Minneapolis, Minnesota 55455
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  • Horacio Bach
    Affiliations
    Department of Microbiology and BioTechnology Institute, University of Minnesota, Minneapolis, Minnesota 55455
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  • David H. Sherman
    Affiliations
    Department of Microbiology and BioTechnology Institute, University of Minnesota, Minneapolis, Minnesota 55455
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  • David C. Lamb
    Affiliations
    Wolfson Laboratory of P-450 Biodiversity, Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales SY23 3DA, United Kingdom
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  • Steven L. Kelly
    Affiliations
    Wolfson Laboratory of P-450 Biodiversity, Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, Wales SY23 3DA, United Kingdom
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  • Michael R. Waterman
    Affiliations
    Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146
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  • Author Footnotes
    * This work was supported by National Institutes of Health Grants GM37942 and ES00267 (to M. R. W.), P30 ES00267 (to L. M. P.), GM48562 (to D. H. S.), by Biotechnology and Biological Sciences Research Council and a Welcome Trust Grant (to S. L. K. and D. C. L.), and by NCI Cancer Biology Training Grant CA09138 (to B. J. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked 舠advertisement舡 in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.The atomic coordinates and the structure factors (code ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Open AccessPublished:January 07, 2003DOI:https://doi.org/10.1074/jbc.M212210200
      Evolutionary links between cytochrome P450 monooxygenases, a superfamily of extraordinarily divergent heme-thiolate proteins catalyzing a wide array of NADPH/NADH- and O2-dependent reactions, are becoming better understood because of availability of an increasing number of fully sequenced genomes. Among other reactions, P450s catalyze the site-specific oxidation of the precursors to macrolide antibiotics in the genus Streptomyces introducing regiochemical diversity into the macrolide ring system, thereby significantly increasing antibiotic activity. Developing effective uses forStreptomyces enzymes in biosynthetic processes and bioremediation requires identification and engineering of additional monooxygenases with activities toward a diverse array of small molecules. To elucidate the molecular basis for substrate specificity of oxidative enzymes toward macrolide antibiotics, the x-ray structure of CYP154C1 from Streptomyces coelicolor A3(2) was determined (Protein Data Bank code 1GWI). Relocation of certain common P450 secondary structure elements, along with a novel structural feature involving an additional ॆ-strand transforming the five-stranded ॆ-sheet into a six-stranded variant, creates an open cleft-shaped substrate-binding site between the two P450 domains. High sequence similarity to macrolide monooxygenases from other microbial species translates into catalytic activity of CYP154C1 toward both 12- and 14-membered ring macrolactonesin vitro.1GWI
      CYP
      cytochrome P450
      MES
      4-morpholineethanesulfonic acid
      The post-genomic era opens new opportunities for structural insight into the evolution of a single protein family within and between species. Cytochrome P450 (CYP)1 monooxygenases are a superfamily of heme-thiolate enzymes that are involved in a wide array of NADPH/NADH- and O2-dependent reactions (
      • Guengerich F.P.
      • MacDonald T.L.
      ). There are currently more than 2000 family members, including a large number of putative P450 open reading frames found in fully sequenced prokaryotic and eukaryotic genomes (drnelson.utmem.edu/CytochromeP450.html). Extensive studies have firmly established their role in the biosynthesis of sterols, fatty acids, and prostaglandins in animals, antibiotics, and other biologically active molecules in bacteria, fungi, and plants as well as in the metabolism of xenobiotic drugs and toxic chemicals (
      • Guengerich F.P.
      ). Accordingly, the extraordinary diversity in amino acid sequence enables wide variation in the substrates utilized and the patterns of oxidation catalyzed by these enzymes.
      A particularly rich source of CYPs is from Streptomycesspp., a group of developmentally complex, Gram-positive bacteria that are known for production of a broad array of biologically active secondary metabolites. Streptomyces coelicolor A3(2) has been investigated extensively as a model system for the study of morphological and physiological development of Streptomycesand for investigation of the genetic control of antibiotic production (
      • Hopwood D.A.
      ). Over the past decade, an increasing number ofStreptomyces spp. have been investigated because of their production of pharmaceutically important compounds including anti-cancer agents, immunosuppressants, and antibiotics. In addition,Streptomyces are being recognized as a source of versatile biocatalysts for the detoxification of hazardous chemicals (
      • Shelton D.R.
      • Khader S.
      • Karns J.S.
      • Pogell B.M.
      ,
      • Jendrossek D.
      • Tomasi G.
      • Kroppenstedt R.M.
      ,
      • Fadullon F.S.
      • Karns J.S.
      • Torrents A.
      ,
      • Obojska A.
      • Lejczak B.
      • Kubrak M.
      ,
      • Gallert C.
      ,
      • Amoroso M.J.
      • Castro G.R.
      • Duran A.
      • Peraud O.
      • Oliver G.
      • Hill R.T.
      ) in bioremediation processes.
      In an effort to analyze fully the complement of CYPs in the industrially important Streptomyces, we decided to focus on the 8.7 Mb S. coelicolor A3(2) genome whose sequence was recently completed by the Sanger Centre (
      • Bentley S.D.
      • Chater K.F.
      • Cerdeno-Tarraga A.M.
      • Challis G.L.
      • Thomson N.R.
      • James K.D.
      • Harris D.E.
      • Quail M.A.
      • Kieser H.
      • Harper D.
      • Bateman A.
      • Brown S.
      • Chandra G.
      • Chen C.W.
      • Collins M.
      • Cronin A.
      • Fraser A.
      • Goble A.
      • Hidalgo J.
      • Hornsby T.
      • Howarth S.
      • Huang C.H.
      • Kieser T.
      • Larke L.
      • Murphy L.
      • Oliver K.
      • O'Neil S.
      • Rabbinowitsch E.
      • Rajandream M.A.
      • Rutherford K.
      • Rutter S.
      • Seeger K.
      • Saunders D.
      • Sharp S.
      • Squares R.
      • Squares S.
      • Taylor K.
      • Warren T.
      • Wietzorrek A.
      • Woodward J.
      • Barrell B.G.
      • Parkhill J.
      • Hopwood D.A.
      ) (www.sanger.ac.uk/Projects/S_coelicolor/). Although 18 CYP open reading frames are found dispersed throughout the S. coelicolorA3(2) chromosome (
      • Bentley S.D.
      • Chater K.F.
      • Cerdeno-Tarraga A.M.
      • Challis G.L.
      • Thomson N.R.
      • James K.D.
      • Harris D.E.
      • Quail M.A.
      • Kieser H.
      • Harper D.
      • Bateman A.
      • Brown S.
      • Chandra G.
      • Chen C.W.
      • Collins M.
      • Cronin A.
      • Fraser A.
      • Goble A.
      • Hidalgo J.
      • Hornsby T.
      • Howarth S.
      • Huang C.H.
      • Kieser T.
      • Larke L.
      • Murphy L.
      • Oliver K.
      • O'Neil S.
      • Rabbinowitsch E.
      • Rajandream M.A.
      • Rutherford K.
      • Rutter S.
      • Seeger K.
      • Saunders D.
      • Sharp S.
      • Squares R.
      • Squares S.
      • Taylor K.
      • Warren T.
      • Wietzorrek A.
      • Woodward J.
      • Barrell B.G.
      • Parkhill J.
      • Hopwood D.A.
      ,
      • Lamb D.C.
      • Skaug T.
      • Song H.-L.
      • Jackson C.J.
      • Podust L.M.
      • Waterman M.R.
      • Kell D.B.
      • Kelly D.E.
      • Kelly S.L.
      ), the functions of the corresponding gene products and their role in various metabolic functions remain largely undefined.
      Most of the currently identified antibiotics are produced by complex biosynthetic systems comprised of clustered gene sets located contiguously on the Streptomyces chromosome (
      • Xue Y.
      • Zhao L.
      • Liu H.W.
      • Sherman D.H.
      ,
      • Bate N.
      • Butler A.R.
      • Gandecha A.R.
      • Cundliffe E.
      ,
      • Fouces R.
      • Mellado E.
      • Diez B.
      • Barredo J.L.
      ,
      • Ikeda H.
      • Nonomiya T.
      • Usami M.
      • Ohta T.
      • Omura S.
      ,
      • Pelzer S.
      • Sussmuth R.
      • Heckmann D.
      • Recktenwald J.
      • Huber P.
      • Jung G.
      • Wohlleben W.
      ,
      • Caffrey P.
      • Lynch S.
      • Flood E.
      • Finnan S.
      • Oliynyk M.
      ,
      • Huang J.
      • Lih C.J.
      • Pan K.H.
      • Cohen S.N.
      ). The clustering of secondary metabolite genes has been an aid in the isolation of P450 monooxygenases involved in antibiotic biosynthesis. Cytochrome P450 monooxygenases are particularly common in polyketide biosynthetic gene clusters, and they catalyze site-specific tailoring reactions leading to the macrolide antibiotics, including methymycin, neomethymycin, and pikromycin (
      • Xue Y.
      • Zhao L.
      • Liu H.W.
      • Sherman D.H.
      ,
      • Xue Y.
      • Wilson D.
      • Zhao L.
      • Liu H.
      • Sherman D.H.
      ,
      • Xue Y.
      • Sherman D.H.
      ,
      • Graziani E.I.
      • Cane D.E.
      • Betlach M.C.
      • Kealey J.T.
      • McDaniel R.
      ,
      • Betlach M.C.
      • Kealey J.T.
      • Ashley G.W.
      • McDaniel R.
      ), novamethymycin (
      • Zhang Q.
      • Sherman D.H.
      ), oleandomycin (
      • Rodriguez A.M.
      • Olano C.
      • Mendez C.
      • Hutchinson C.R.
      • Salas J.A.
      ), amphotericin (
      • Caffrey P.
      • Lynch S.
      • Flood E.
      • Finnan S.
      • Oliynyk M.
      ), and erythromycin (
      • Haydock S.F.
      • Dowson J.A.
      • Dhillon N.
      • Roberts G.A.
      • Cortes J.
      • Leadlay P.F.
      ,
      • Weber J.M.
      • Leung J.O.
      • Swanson S.J.
      • Idler K.B.
      • McAlpine J.B.
      ). Additionally, CYPs are involved in the formation of the anticancer agent epothilone (
      • Molnar I.
      • Schupp T.
      • Ono M.
      • Zirkle R.
      • Milnamow M.
      • Nowak-Thompson B.
      • Engel N.
      • Toupet C.
      • Stratmann A.
      • Cyr D.D.
      • Gorlach J.
      • Mayo J.M.
      • Hu A.
      • Goff S.
      • Schmid J.
      • Ligon J.M.
      ,
      • Tang L.
      • Shah S.
      • Chung L.
      • Carney J.
      • Katz L.
      • Khosla C.
      • Julien B.
      ), immunosuppressant rapamycin (
      • Molnar I.
      • Aparicio J.F.
      • Haydock S.F.
      • Khaw L.E.
      • Schwecke T.
      • Konig A.
      • Staunton J.
      • Leadlay P.F.
      ,
      • Chung L.
      • Liu L.
      • Patel S.
      • Carney J.R.
      • Reeves C.D.
      ), the growth promoter tylosin (
      • Fouces R.
      • Mellado E.
      • Diez B.
      • Barredo J.L.
      ), and the antiparasitic agent avermectin (
      • Ikeda H.
      • Nonomiya T.
      • Usami M.
      • Ohta T.
      • Omura S.
      ). These reactions typically occur during the late stages of biosynthesis after formation of the core ring system by a polyketide synthase. The hydroxyl or epoxide substituents provide an important layer of structural variability into the final natural product structures and often significantly influence biological activity (
      • Xue Y.
      • Zhao L.
      • Liu H.W.
      • Sherman D.H.
      ,
      • Betlach M.C.
      • Kealey J.T.
      • Ashley G.W.
      • McDaniel R.
      ). P450 monooxygenases are also involved in one of the initial steps in formation of the coumarin group of antibiotics (
      • Chen H.
      • Walsh C.T.
      ), and of the peptidyl nucleoside antibiotic nikkomycin (
      • Lauer B.
      • Russwurm R.
      • Schwarz W.
      • Kalmanczhelyi A.
      • Bruntner C.
      • Rosemeier A.
      • Bormann C.
      ), as well as in oxidative tailoring of the vancomycin-like glycopeptides balhimycin (
      • Pelzer S.
      • Sussmuth R.
      • Heckmann D.
      • Recktenwald J.
      • Huber P.
      • Jung G.
      • Wohlleben W.
      ) and complestatin (
      • Chiu H.T.
      • Hubbard B.K.
      • Shah A.N.
      • Eide J.
      • Fredenburg R.A.
      • Walsh C.T.
      • Khosla C.
      ). Ultimately, the power to manipulate macrolide metabolic systems using combinatorial biosynthetic technology (
      • Carreras C.W.
      • Santi D.V.
      ,
      • Hutchinson C.R.
      ,
      • Yoon Y.J.
      • Beck B.J.
      • Kim B.S.
      • Kang H.Y.
      • Reynolds K.A.
      • Sherman D.H.
      ) will be extended by identification and/or engineering of additional monooxygenases with versatile activities to provide novel biologically active natural products.
      Our current study was motivated by the intriguing amino acid sequence relationship between a number of S. coelicolor A3(2) CYPs that show significant similarity with P450 monooxygenases from other microorganisms involved in regio-specific oxidation of macrolide antibiotics. Among eight crystal structures reported for cytochrome P450 over the last 17 years, only one has been reported for a macrolide hydroxylase (P450 EryF from Saccharopolyspora erythraea) (
      • Cupp-Vickery J.R.
      • Poulos T.L.
      ). EryF is involved in hydroxylation of C-6 of the 14-membered ring macrolactone 6-deoxyerythronolide B in the erythromycin biosynthetic pathway. To elucidate structural, functional, and evolutionary aspects of monooxygenases that tailor macrolide and xenobiotic molecules, crystallographic analysis of the cytochrome P450 complement of S. coelicolor A3(2) (
      • Bentley S.D.
      • Chater K.F.
      • Cerdeno-Tarraga A.M.
      • Challis G.L.
      • Thomson N.R.
      • James K.D.
      • Harris D.E.
      • Quail M.A.
      • Kieser H.
      • Harper D.
      • Bateman A.
      • Brown S.
      • Chandra G.
      • Chen C.W.
      • Collins M.
      • Cronin A.
      • Fraser A.
      • Goble A.
      • Hidalgo J.
      • Hornsby T.
      • Howarth S.
      • Huang C.H.
      • Kieser T.
      • Larke L.
      • Murphy L.
      • Oliver K.
      • O'Neil S.
      • Rabbinowitsch E.
      • Rajandream M.A.
      • Rutherford K.
      • Rutter S.
      • Seeger K.
      • Saunders D.
      • Sharp S.
      • Squares R.
      • Squares S.
      • Taylor K.
      • Warren T.
      • Wietzorrek A.
      • Woodward J.
      • Barrell B.G.
      • Parkhill J.
      • Hopwood D.A.
      ,
      • Lamb D.C.
      • Skaug T.
      • Song H.-L.
      • Jackson C.J.
      • Podust L.M.
      • Waterman M.R.
      • Kell D.B.
      • Kelly D.E.
      • Kelly S.L.
      ) was initiated. We report here the first structural and functional analysis of the monooxygenase CYP154C1 from this organism determined to 1.92 Å resolution.

      Acknowledgments

      We thank Jarrod A. Smith and the Vanderbilt University Center for Structural Biology computing facilities for expert technical assistance, Dr. Stanley N. Cohen and Dr. Jianqiang Huang (Stanford University) for providing microarray information on expression of individual CYP genes, and Dr. Thomas Poulos (University of California, Irvine) for interest in the work, helpful discussions, and critical reading of the manuscript.

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