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Characterization of a ring-hydroxylating dioxygenase from phenanthrene-degrading Sphingomonas sp. strain LH128 able to oxidize benz[a]anthracene

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Abstract

Sphingomonas sp. strain LH128 was isolated from a polycyclic aromatic hydrocarbon (PAH)-contaminated soil using phenanthrene as the sole source of carbon and energy. A dioxygenase complex, phnA1fA2f, encoding the α and β subunit of a terminal dioxygenase responsible for the initial attack on PAHs, was identified and isolated from this strain. PhnA1f showed 98%, 78%, and 78% identity to the α subunit of PAH dioxygenase from Novosphingobium aromaticivorans strain F199, Sphingomonas sp. strain CHY-1, and Sphingobium yanoikuyae strain B1, respectively. When overexpressed in Escherichia coli, PhnA1fA2f was able to oxidize low-molecular-weight PAHs, chlorinated biphenyls, dibenzo-p-dioxin, and the high-molecular-weight PAHs benz[a]anthracene, chrysene, and pyrene. The action of PhnA1fA2f on benz[a]anthracene produced two benz[a]anthracene dihydrodiols.

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References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Armengaud J, Happe B, Timmis KN (1998) Genetic analysis of dioxin dioxygenase of Sphingomonas sp. strain RW1: catabolic genes dispersed on the genome. J Bacteriol 180:3954–3966

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bastiaens L, Springael D, Wattiau P, Harms H, deWachter R, Verachtert H, Diels L (2000) Isolation of adherent polycyclic aromatic hydrocarbon (PAH)-degrading bacteria using PAH-sorbing carriers. Appl Environ Microbiol 66:1834–1843

    CAS  PubMed  PubMed Central  Google Scholar 

  • Boyd DR, Sharma ND, Allen CCR (2001) Aromatic dioxygenases: molecular biocatalysis and applications. Curr Opin Biotechnol 12:564–573

    CAS  PubMed  Google Scholar 

  • Boyd DR, Sharma ND, Sbircea L, Murphy D, Belhocine T, Malone JF, James SL, Allen CCR, Hamilton JTG (2008) Azaarene cis-dihydrodiol-derived 2,2′-bipyridine ligands for asymmetric allylic oxidation and cyclopropanation. Chem Commun 43:5535–5537

    Google Scholar 

  • Bunz PV, Cook AM (1993) Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system. J Bacteriol 175:6467–6475

    CAS  PubMed  PubMed Central  Google Scholar 

  • Butler CS, Mason JR (1997) Structure–function analysis of the bacterial aromatic ring-hydroxylating dioxygenases. Adv Microb Physiol 38:47–84

    CAS  PubMed  Google Scholar 

  • Cai M, Xun L (2002) Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723. J Bacteriol 184:4672–4680

    CAS  PubMed  PubMed Central  Google Scholar 

  • Cerniglia CE (1992) Biodegradation of polycyclic aromatic hydrocarbons. Biodegradation 3:351–368

    CAS  Google Scholar 

  • de Lorenzo V, Eltis L, Kessler B, Timmis KN (1993) Analysis of Pseudomonas gene products using lacI q/Ptrp-lac plasmids and transposons that confer conditional phenotypes. Gene 123:17–24

    PubMed  Google Scholar 

  • Demaneche S, Meyer C, Micoud J, Louwagie M, Willison JC, Jouanneau Y (2004) Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a Sphingomonas strain that degrades various polycyclic aromatic hydrocarbons. Appl Environ Microbiol 70:6714–6725

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dimitriou-Christidis P, Autenrieth RL, McDonald TJ, Desai AM (2007) Measurement of biodegradability parameters for single unsubstituted and methylated polycyclic aromatic hydrocarbons in liquid bacterial suspensions. Biotechnol Bioeng 97:922–932

    CAS  PubMed  Google Scholar 

  • Feng X, Ou LT, Ogram A (1997) Plasmid-mediated mineralization of carbofuran by Sphingomonas sp. strain CF06. Appl Environ Microbiol 63:1332–1337

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ferraro DJ, Gakhar L, Ramaswamy S (2005) Rieske business: structure–function of rieske non-heme oxygenases. Biochem Biophys Res Commun 338:175–190

    CAS  PubMed  Google Scholar 

  • Fortnagel P, Harms H, Wittich RM, Krohn S, Meyer H, Sinnwell V, Wilkes H, Francke W (1990) Metabolism of dibenzofuran by Pseudomonas sp. strain HH69 and the mixed culture HH27. Appl Environ Microbiol 56:1148–1156

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fujii K, Satomi M, Morita N, Motomura T, Tanaka T, Kikuchi S (2003) Novosphingobium tardaugens sp. nov., an oestradiol-degrading bacterium isolated from activated sludge of a sewage treatment plant in Tokyo. Int J Syst Evol Microbiol 53:47–52

    CAS  PubMed  Google Scholar 

  • Furukawa K, Suenaga H, Goto M (2004) Biphenyl dioxygenases: functional versatilities and directed evolution. J Bacteriol 186:5189–5196

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gakhar L, Malik ZA, Allen CCR, Lipscomb DA, Larkin MJ, Ramaswamy S (2005) Structure and increased thermostability of Rhodococcus sp. naphthalene 1,2-dioxygenase. J Bacteriol 197:7222–7231

    Google Scholar 

  • Happe B, Eltis LD, Poth H, Hedderich R, Timmis KN (1993) Characterization of 2,2′,3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1. J Bacteriol 175:7313–7320

    CAS  PubMed  PubMed Central  Google Scholar 

  • Hong HB, Chang YS, Nam IH, Fortnagel P, Schmidt S (2002) Biotransformation of 2,7-dichloro- and 1,2,3,4-tetrachlorodibenzo-p-dioxin by Sphingomonas wittichii RW1. Appl Environ Microbiol 68:2584–2588

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jakoncic J, Jouanneau Y, Meyer C, Stojanoff V (2007a) The catalytic pocket of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1. Biochem Biophys Res Commun 352:861–866

    CAS  PubMed  Google Scholar 

  • Jakoncic J, Jouanneau Y, Meyer C, Stojanoff V (2007b) The crystal structure of the ring-hydroxylating dioxygenase from Sphingomonas CHY-1. FEBS J 274:2470–2481

    CAS  PubMed  Google Scholar 

  • Johannesen H, Sorensen SR, Aamand J (2003) Mineralization of soil-aged isoproturon and isoproturon metabolites by Sphingomonas sp. strain SRS2. J Environ Qual 32:1250–1257

    CAS  PubMed  Google Scholar 

  • Johnsen AR, Wick LY, Harms H (2005) Principles of microbial PAH-degradation in soil. Environ Pollut 133:71–84

    CAS  PubMed  Google Scholar 

  • Jouanneau Y, Meyer C, Jakoncic J, Stojanoff V, Gaillard J (2006) Characterization of a naphthalene dioxygenase endowed with an exceptionally broad substrate specificity toward polycyclic aromatic hydrocarbons. Biochemistry 45:12380–12391

    CAS  PubMed  Google Scholar 

  • Kauppi B, Lee K, Carredano E, Parales RE, Gibson DT, Eklund H, Ramaswamy S (1998) Structure of an aromatic-ring-hydroxylating dioxygenase-naphthalene 1,2-dioxygenase. Structure 6:571–586

    CAS  PubMed  Google Scholar 

  • Kim E, Zylstra GJ (1999) Functional analysis of genes involved in biphenyl, naphthalene, phenanthrene, and m-xylene degradation by Sphingomonas yanoikuyae B1. J Ind Microbiol Biotechnol 23:294–302

    PubMed  Google Scholar 

  • Kim S, Chun J, Bae K, Kim Y (2000) Polyphasic assignment of an aromatic-degrading Pseudomonas sp., strain DJ77, in the genus Sphingomonas as Sphingomonas chungbukensis sp. nov. Int J Syst Evol Microbiol 50:1641–1647

    CAS  PubMed  Google Scholar 

  • Kim IS, Ryu JY, Hur HG, Gu MB, Kim SD, Shim JH (2004) Sphingomonas sp. strain SB5 degrades carbofuran to a new metabolite by hydrolysis at the furanyl ring. J Agric Food Chem 52:2309–2314

    CAS  PubMed  Google Scholar 

  • Krivobok S, Kuony S, Meyer C, Louwagie M, Willison JC, Jouanneau Y (2003) Identification of pyrene-induced proteins in Mycobacterium sp. strain 6PY1: evidence for two ring-hydroxylating dioxygenases. J Bacteriol 185:3828–3841

    CAS  PubMed  PubMed Central  Google Scholar 

  • Leys NM, Ryngaert A, Bastiaens L, Verstraete W, Top EM, Springael D (2004) Occurrence and phylogenetic diversity of Sphingomonas strains in soils contaminated with polycyclic aromatic hydrocarbons. Appl Environ Microbiol 70:1944–1955

    CAS  PubMed  PubMed Central  Google Scholar 

  • McIver AM, Janardhan Garikipati SVB, Bankole KS, Gyamerah M, Peeples TL (2008) Microbial oxidation of naphthalene to cis-1,2-naphthalene dihydrodiol using naphthalene dioxygenase in biphasic media. Biotechnol Prog 24:593–598

    CAS  PubMed  Google Scholar 

  • Mergeay M, Nies D, Schlegel HG, Gerits J, Charles P, Van Gijsegem F (1985) Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals. J Bacteriol 162:328–334

    CAS  PubMed  PubMed Central  Google Scholar 

  • Ni Chadhain SM, Moritz EM, Kim E, Zylstra GJ (2007) Identification, cloning, and characterization of a multicomponent biphenyl dioxygenase from Sphingobium yanoikuyae strain B1. J Ind Microbiol Biotechnol 34:605–613

    Google Scholar 

  • Parales RE (2003) The role of active-site residues in naphthalene dioxygenase. J Ind Microbiol Biotechnol 30:271–278

    CAS  PubMed  Google Scholar 

  • Parales RE, Parales JV, Gibson DT (1999) Aspartate 205 in the catalytic domain of naphthalene dioxygenase is essential for activity. J Bacteriol 181:1831–1837

    CAS  PubMed  PubMed Central  Google Scholar 

  • Parales RE, Lee K, Resnick SM, Jiang H, Lessner DJ, Gibson DT (2000a) Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. J Bacteriol 182:1641–1649

    CAS  PubMed  PubMed Central  Google Scholar 

  • Parales RE, Resnick SM, Yu CL, Boyd DR, Sharma ND, Gibson DT (2000b) Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit. J Bacteriol 182:5495–5504

    CAS  PubMed  PubMed Central  Google Scholar 

  • Peng X, Masai E, Kitayama H, Harada K, Katayama Y, Fukuda M (2002) Characterization of the 5-carboxyvanillate decarboxylase gene and its role in lignin-related biphenyl catabolism in Sphingomonas paucimobilis SYK-6. Appl Environ Microbiol 68:4407–4415

    CAS  PubMed  PubMed Central  Google Scholar 

  • Pinyakong O, Habe H, Supaka N, Pinpanichkarn P, Juntongjin K, Yoshida T, Furihata K, Nojiri H, Yamane H, Omori T (2000) Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2. FEMS Microbiol Lett 191:115–121

    CAS  PubMed  Google Scholar 

  • Pinyakong O, Habe H, Omori T (2003a) The unique aromatic catabolic genes in sphingomonads degrading polycyclic aromatic hydrocarbons (PAHs). J Gen Appl Microbiol 49:1–19

    CAS  PubMed  Google Scholar 

  • Pinyakong O, Habe H, Yoshida T, Nojiri H, Omori T (2003b) Identification of three novel salicylate 1-hydroxylases involved in the phenanthrene degradation of Sphingobium sp. strain P2. Biochem Biophys Res Commun 301:350–357

    CAS  PubMed  Google Scholar 

  • Resnick SM, Torok DS, Gibson DT (1993) Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase. FEMS Microbiol Lett 113:297–302

    CAS  PubMed  Google Scholar 

  • Romine MF, Stillwell LC, Wong KK, Thurston SJ, Sisk EC, Sensen C, Gaasterland T, Fredrickson JK, Saffer JD (1999) Complete sequence of a 184-kilobase catabolic plasmid from Sphingomonas aromaticivorans F199. J Bacteriol 181:1585–1602

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1990) Molecular cloning: a laboratory manual. Cold Spring Laboratory Harbor Press, Cold Spring Harbor, NY

    Google Scholar 

  • Schuler L, Ni Chadhain SM, Jouanneau Y, Meyer C, Zylstra GL, Hols P, Agathos SN (2008) Characterization of a novel angular dioxygenase from fluorene-degrading Sphingomonas sp. strain LB126. Appl Environ Microbiol 74:1050–1057

    CAS  PubMed  Google Scholar 

  • Sorensen SR, Ronen Z, Aamand J (2001) Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon. Appl Environ Microbiol 67:5403–5409

    CAS  PubMed  PubMed Central  Google Scholar 

  • Stolz A (1999) Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6. J Ind Microbiol Biotechnol 23:391–399

    CAS  PubMed  Google Scholar 

  • Story SP, Parker SH, Hayasaka SS, Riley MB, Kline EL (2001) Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505. J Ind Microbiol Biotechnol 26:369–382

    CAS  PubMed  Google Scholar 

  • Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25:4876–4882

    CAS  PubMed  PubMed Central  Google Scholar 

  • van Herwijnen R, de Graaf C, Govers HA, Parsons JR (2004) Estimation of kinetic parameter for the biotransformation of three-ring azaarenes by the phenanthrene-degrading strain Sphingomonas sp. LH128. Environ Toxicol Chem 23:331–338

    PubMed  Google Scholar 

  • Wackett LP (2002) Mechanism and application of Rieske non-heme iron dioxygenases. Enzyme Microb Technol 31:577–587

    CAS  Google Scholar 

  • Willison JC (2004) Isolation and characterization of a novel sphingomonad capable of growth with chrysene as sole carbon and energy source. FEMS Microbiol Lett 241:143–150

    CAS  PubMed  Google Scholar 

  • Yrjala K, Paulin L, Kilpi S, Romantschuk M (1994) Cloning of cmpE, a plasmid-borne catechol 2,3-dioxygenase-encoding gene from the aromatic- and chloroaromatic-degrading Pseudomonas sp. HV3. Gene 138:119–121

    CAS  PubMed  Google Scholar 

  • Yu CL, Liu W, Ferraro DJ, Brown EN, Parales JV, Ramaswamy S, Zylstra GJ, Gibson DT, Parales RE (2007) Purification, characterization, and crystallization of the components of a biphenyl dioxygenase system from Sphingobium yanoikuyae B1. J Ind Microbiol Biotechnol 34:311–324

    CAS  PubMed  Google Scholar 

  • Zielinski M, Kahl S, Hecht HJ, Hofer B (2003) Pinpointing biphenyl dioxygenase residues that are crucial for substrate interaction. J Bacteriol 185:6976–6980

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zielinski M, Kahl S, Standfuss-Gabisch C, Camara B, Seeger M, Hofer B (2006) Generation of novel-substrate-accepting biphenyl dioxygenases through segmental random mutagenesis and identification of residues involved in enzyme specificity. Appl Environ Microbiol 72:2191–2199

    CAS  PubMed  PubMed Central  Google Scholar 

  • Zylstra GJ, Kim E (1997) Aromatic hydrocarbon degradation by Sphingomonas yanoikuyae B1. J Ind Microbiol Biotechnol 19:408–414

    CAS  PubMed  Google Scholar 

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Acknowledgements

L.S. gratefully acknowledges the Fund for the Promotion of Research in Industry and Agriculture (F.R.I.A.), Belgium, for providing a doctoral fellowship. L.S. also wishes to thank the members of the Unit of Physiological Biochemistry (FYSA), Catholic University of Louvain, for their daily help and constructive remarks for many years. PH is a research associate at the Belgian National Fund for Scientific Research (FNRS).

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Authors and Affiliations

  1. Unité de Génie Biologique, Institut des Sciences de la Vie, Université Catholique de Louvain, Place Croix du Sud, 2/19, 1348, Louvain-la-Neuve, Belgium

    Luc Schuler, Maria Pouli & Spiros N. Agathos

  2. CEA, iRTSV, Laboratoire de Chimie et Biologie des Métaux and UMR 5249 CNRS, Université J. Fourier, 38054, Grenoble Cedex 9, France

    Yves Jouanneau & Christine Meyer

  3. Biotechnology Center for Agriculture and the Environment, Cook College, Rutgers University, New Brunswick, NJ, 08901-8520, USA

    Sinéad M. Ní Chadhain & Gerben J. Zylstra

  4. Unité de Génétique, Institut des Sciences de la Vie, Université Catholique de Louvain, Place Croix du Sud 5, 1348, Louvain-la-Neuve, Belgium

    Pascal Hols

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  1. Luc Schuler
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Schuler, L., Jouanneau, Y., Ní Chadhain, S.M. et al. Characterization of a ring-hydroxylating dioxygenase from phenanthrene-degrading Sphingomonas sp. strain LH128 able to oxidize benz[a]anthracene. Appl Microbiol Biotechnol 83, 465–475 (2009). https://doi.org/10.1007/s00253-009-1858-2

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