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Biochemical Studies and Ligand-bound Structures of Biphenyl Dehydrogenase from Pandoraea pnomenusa Strain B-356 Reveal a Basis for Broad Specificity of the Enzyme*

Open AccessPublished:August 31, 2011DOI:https://doi.org/10.1074/jbc.M111.291013
      Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphBB-356), the crystal structures of the apo-enzyme, the binary complex with NAD+, and the ternary complexes with NAD+-2,3-dihydroxybiphenyl and NAD+-4,4′-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphBB-356 converts the dihydrodiol metabolites of 3,3′-dichlorobiphenyl, 2,4,4′-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphBB-356 elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.

      Introduction

      Aerobic biodegradation of polychlorinated biphenyls (PCBs)
      The abbreviations used are: PCB
      polychlorinated biphenyl
      23-DB
      2,3-dihydroxybiphenyl
      44-DB
      4,4′-dihydroxybiphenyl
      SDR
      short-chain dehydrogenase/oxidoreductase
      PDB
      Protein Data Bank
      r.m.s.
      root mean square
      BphB
      cis-2,3-dihydro-2,3-dihydroxybiphenyl dehydrogenase
      BphC
      2,3-dihydroxybiphenyl 1,2-dioxygenase
      BphD
      2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase
      BPDD
      cis-(2R,3S)-dihydroxy-1-phenyl-cyclohexa-4,6-diene
      BPDO
      biphenyl dioxygenase.
      by bacteria occurs through an oxidative process (
      • Strand S.E.
      ,
      • Furukawa K.
      ,
      • Ohtsubo Y.
      • Kudo T.
      • Tsuda M.
      • Nagata Y.
      ,
      • Borja J.
      • Taleon D.M.
      • Auresenia J.
      • Gallardo S.
      ,
      • Pieper D.H.
      ). The biphenyl degrading pathway, encoded by the bph operon, metabolizes several PCB congeners to the corresponding chlorobenzoates (
      • Furukawa K.
      • Fujihara H.
      ). This pathway has been found in many bacteria among which Pandoraea pnomenusa strain B-356, Burkholderia xenovorans strain LB400, Pseudomonas pseudoalcaligenes strain KF707, and Pseudomonas sp. strain KKS102 have been thoroughly investigated (
      • Fukuda M.
      • Yasukochi Y.
      • Kikuchi Y.
      • Nagata Y.
      • Kimbara K.
      • Horiuchi H.
      • Takagi M.
      • Yano K.
      ,
      • Haddock J.D.
      • Nadim L.M.
      • Gibson D.T.
      ,
      • Hurtubise Y.
      • Barriault D.
      • Powlowski J.
      • Sylvestre M.
      ,
      • Pieper D.H.
      • Reineke W.
      ,
      • Taira K.
      • Hirose J.
      • Hayashida S.
      • Furukawa K.
      ). The bph pathway comprises four enzymes that in P. pnomenusa B-356 are encoded by bphAEFGBCD. The biphenyl dioxygenase system (BphAEFG) catalyzes the first reaction, the insertion of two oxygen atoms into vicinal ortho-meta carbons of biphenyl to generate cis-2,3-dihydro-2,3-dihydroxybiphenyl. cis-2,3-Dihydro-2,3-dihydroxybiphenyl dehydrogenase (BphB), the second enzyme of this pathway, catalyzes a dehydrogenation reaction to produce 2,3-dihydroxybiphenyl. 2,3-Dihydroxybiphenyl 1,2-dioxygenase (BphC) and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD) then sequentially metabolize 2,3-dihydroxybiphenyl to chlorobenzoate and 2-oxo-4-pentadienoate as shown in Fig. 1 (
      • Ohtsubo Y.
      • Kudo T.
      • Tsuda M.
      • Nagata Y.
      ,
      • Pieper D.H.
      ).
      Figure thumbnail gr1
      FIGURE 1Schematic representation of aerobic degradation by the biphenyl/polychlorinated degradation catabolic pathway.
      Many investigations have shown that the PCB-degrading ability differs among the different PCB-degrading bacteria (
      • Furukawa K.
      ,
      • Furukawa K.
      • Fujihara H.
      ,
      • Pieper D.H.
      • Reineke W.
      ). The biphenyl dioxygenase system critically determines which of the 209 PCB congeners can be metabolized by each strain. For example, a recent study showed that the biphenyl dioxygenase of strain B-356 metabolizes 2,6-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,4,4′-trichlorobiphenyl, whereas the biphenyl dioxygenase of strain LB400 metabolizes these congeners poorly. On the other hand, strain BPDOB-356 is unable to catalyze the meta-para oxygenation of 2,2′,5,5′-tetrachlorobiphenyl, whereas BPDOLB400 catalyzes it efficiently. The versatility of BphB toward BPDO metabolites of PCB congeners has not been thoroughly investigated (
      • Gómez-Gil L.
      • Kumar P.
      • Barriault D.
      • Bolin J.T.
      • Sylvestre M.
      • Eltis L.D.
      ). A previous report, however, showed that both BphBLB400 and BphBB-356 are able to oxidize 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl, the product resulting from the metabolism of 2,2′,5,5′-tetrachlorobiphenyl by BPDOLB400 (
      • Barriault D.
      • Vedadi M.
      • Powlowski J.
      • Sylvestre M.
      ). To the best of our knowledge, the ability of BphBB-356 to metabolize the product generated from 2,6-dichlorobiphenyl or 2,4,4′-trichlorobiphenyl has never been examined. But recently it has been shown that the dihydrodiol dehydrogenase from Sphingomonas sp. strain CHY-1 can oxidize a wide range of polyaromatic hydrocarbon dihydrodiols (
      • Jouanneau Y.
      • Meyer C.
      ).
      BphB occurs as a homotetramer comprised of 29.4-kDa subunits. It is an NAD+-dependent oxidoreductase, related to other cis-dihydrodiol dehydrogenases involved in aromatic degradation pathways and belongs to a very large family of short-chain dehydrogenase (the SDR family) (
      • Jörnvall H.
      • Persson B.
      • Krook M.
      • Atrian S.
      • Gonzàlez-Duarte R.
      • Jeffery J.
      • Ghosh D.
      ,
      • Sylvestre M.
      • Hurtubise Y.
      • Barriault D.
      • Bergeron J.
      • Ahmad D.
      ). Although, the crystal structure of the NAD+-bound form of BphBLB400 is known (
      • Hülsmeyer M.
      • Hecht H.J.
      • Niefind K.
      • Hofer B.
      • Eltis L.D.
      • Timmis K.N.
      • Schomburg D.
      ), there is no structure available with bound substrate, product, or any product analogs. Furthermore, the substrate binding loop in the published structure of BphBLB400 is disordered (
      • Hülsmeyer M.
      • Hecht H.J.
      • Niefind K.
      • Hofer B.
      • Eltis L.D.
      • Timmis K.N.
      • Schomburg D.
      ). Therefore, the structural features involved in substrate binding of the cis-dihydrodiol dehydrogenases from the aromatic degradation pathways are still undetermined.
      To get more insight into the binding mode of the ligand with BphB, we have compared the crystal structure of the apo form of BphBB-356 with that of its NAD+-bound form (binary state) or of its ternary complex with its coenzyme-NAD+ and its product (2,3-dihydroxybiphenyl) or a product analog (4,4′-dihydroxybiphenyl). In addition, we also describe a structure showing an intermediate state of the substrate binding loop. These three-dimensional structures provide insight to the binding mode of ligand with the enzyme. We were able to identify the series of conformational changes in the substrate binding loop that occur during ligand binding. Additionally, our docking studies are consistent with the biochemical experiments that examine the ability of BphBB-356 to metabolize and accommodate a large range of chlorinated substrates.

      Acknowledgments

      We thank the Macromolecular Crystallographic Facility (MCU) at the Indian Institute of Technology, IIT Roorkee. We also thank the Department of Biotechnology (DBT), India, for providing financial assistance and the allocation and provision of synchrotron beamtime at the BM14, ESRF (Grenoble, France). We thank Hassan Belrhali and Babu Manjasetty for help during data collection at the synchrotron. We are grateful to Dr. David Neau for carefully reading the manuscript and Dr. Ashwani Kumar Sharma for helpful discussions.

      References

        • Strand S.E.
        CEWA, ESC, MICRO. 2004; 518: 1-10
        • Furukawa K.
        Trends Biotechnol. 2003; 21: 187-190
        • Ohtsubo Y.
        • Kudo T.
        • Tsuda M.
        • Nagata Y.
        Appl. Microbiol. Biotechnol. 2004; 65: 250-258
        • Borja J.
        • Taleon D.M.
        • Auresenia J.
        • Gallardo S.
        Process Biochem. 2005; 40: 1999-2013
        • Pieper D.H.
        Appl. Microbiol. Biotechnol. 2005; 67: 170-191
        • Furukawa K.
        • Fujihara H.
        J. Biosci. Bioeng. 2008; 105: 433-449
        • Fukuda M.
        • Yasukochi Y.
        • Kikuchi Y.
        • Nagata Y.
        • Kimbara K.
        • Horiuchi H.
        • Takagi M.
        • Yano K.
        Biochem. Biophys. Res. Commun. 1994; 202: 850-856
        • Haddock J.D.
        • Nadim L.M.
        • Gibson D.T.
        J. Bacteriol. 1993; 175: 395-400
        • Hurtubise Y.
        • Barriault D.
        • Powlowski J.
        • Sylvestre M.
        J. Bacteriol. 1995; 177: 6610-6618
        • Pieper D.H.
        • Reineke W.
        Curr. Opin. Biotechnol. 2000; 11: 262-270
        • Taira K.
        • Hirose J.
        • Hayashida S.
        • Furukawa K.
        J. Biol. Chem. 1992; 267: 4844-4853
        • Gómez-Gil L.
        • Kumar P.
        • Barriault D.
        • Bolin J.T.
        • Sylvestre M.
        • Eltis L.D.
        J. Bacteriol. 2007; 189: 5705-5715
        • Barriault D.
        • Vedadi M.
        • Powlowski J.
        • Sylvestre M.
        Biochem. Biophys. Res. Commun. 1999; 260: 181-187
        • Jouanneau Y.
        • Meyer C.
        Appl. Environ. Microbiol. 2006; 72: 4726-4734
        • Jörnvall H.
        • Persson B.
        • Krook M.
        • Atrian S.
        • Gonzàlez-Duarte R.
        • Jeffery J.
        • Ghosh D.
        Biochemistry. 1995; 34: 6003-6013
        • Sylvestre M.
        • Hurtubise Y.
        • Barriault D.
        • Bergeron J.
        • Ahmad D.
        Appl. Environ. Microbiol. 1996; 62: 2710-2715
        • Hülsmeyer M.
        • Hecht H.J.
        • Niefind K.
        • Hofer B.
        • Eltis L.D.
        • Timmis K.N.
        • Schomburg D.
        Protein Sci. 1998; 7: 1286-1293
        • Patil D.N.
        • Tomar S.
        • Sylvestre M.
        • Kumar P.
        Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2010; 66: 1517-1520
        • Otwinowski Z.
        • Minor W.
        Methods Enzymol. 1997; 276: 307-326
        • Collaborative Computational Project, N
        Acta Crystallogr. 1994; 50: 760-763
        • Berman H.M.
        • Battistuz T.
        • Bhat T.N.
        • Bluhm W.F.
        • Bourne P.E.
        • Burkhardt K.
        • Feng Z.
        • Gilliland G.L.
        • Iype L.
        • Jain S.
        • Fagan P.
        • Marvin J.
        • Padilla D.
        • Ravichandran V.
        • Schneider B.
        • Thanki N.
        • Weissig H.
        • Westbrook J.D.
        • Zardecki C.
        Acta Crystallogr. D Biol. Crystallogr. 2002; 58: 899-907
        • Brünger A.T.
        • Adams P.D.
        • Clore G.M.
        • DeLano W.L.
        • Gros P.
        • Grosse-Kunstleve R.W.
        • Jiang J.S.
        • Kuszewski J.
        • Nilges M.
        • Pannu N.S.
        • Read R.J.
        • Rice L.M.
        • Simonson T.
        • Warren G.L.
        Acta Crystallogr. D Biol. Crystallogr. 1998; 54: 905-921
        • Murshudov G.N.
        • Vagin A.A.
        • Dodson E.J.
        Acta Crystallogr. D Biol. Crystallogr. 1997; 53: 240-255
        • Emsley P.
        • Cowtan K.
        Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132
        • Laskowski R.A.
        • MacArthur M.W.
        • Moss D.S.
        • Thornton J.M.
        J. Appl. Crystallogr. 1993; 26: 283-291
        • DeLano W.L.
        The PyMOL Molecular Graphics System. Schrödinger, LLC, New York2002
        • Vedadi M.
        • Barriault D.
        • Sylvestre M.
        • Powlowski J.
        Biochemistry. 2000; 39: 5028-5034
        • Friesner R.A.
        • Murphy R.B.
        • Repasky M.P.
        • Frye L.L.
        • Greenwood J.R.
        • Halgren T.A.
        • Sanschagrin P.C.
        • Mainz D.T.
        J. Med. Chem. 2006; 49: 6177-6196
        • Barriault D.
        • Plante M.M.
        • Sylvestre M.
        J. Bacteriol. 2002; 184: 3794-3800
        • Rossmann M.G.
        • Adams M.J.
        • Buehner M.
        • Ford G.C.
        • Hackert M.L.
        • Liljas A.
        • Rao S.T.
        • Banaszak L.J.
        • Hill E.
        • Tsernoglou D.
        • Webb L.
        J. Mol. Biol. 1973; 76: 533-537
        • Tanaka N.
        • Nonaka T.
        • Nakamura K.T.
        • Hara A.
        Curr. Org. Chem. 2001; 5: 89-111
        • Tanaka N.
        • Nonaka T.
        • Tanabe T.
        • Yoshimoto T.
        • Tsuru D.
        • Mitsui Y.
        Biochemistry. 1996; 35: 7715-7730
        • Ito K.
        • Nakajima Y.
        • Ichihara E.
        • Ogawa K.
        • Katayama N.
        • Nakashima K.
        • Yoshimoto T.
        J. Mol. Biol. 2006; 355: 722-733
        • Andersson A.
        • Jordan D.
        • Schneider G.
        • Lindqvist Y.
        FEBS Lett. 1997; 400: 173-176
        • Hosfield D.J.
        • Wu Y.
        • Skene R.J.
        • Hilgers M.
        • Jennings A.
        • Snell G.P.
        • Aertgeerts K.
        J. Biol. Chem. 2005; 280: 4639-4648
        • Filling C.
        • Berndt K.D.
        • Benach J.
        • Knapp S.
        • Prozorovski T.
        • Nordling E.
        • Ladenstein R.
        • Jörnvall H.
        • Oppermann U.
        J. Biol. Chem. 2002; 277: 25677-25684
        • Oppermann U.
        • Filling C.
        • Hult M.
        • Shafqat N.
        • Wu X.
        • Lindh M.
        • Shafqat J.
        • Nordling E.
        • Kallberg Y.
        • Persson B.
        • Jörnvall H.
        Chem. Biol. Interact. 2003; 143: 247-253
        • Holm L.
        • Rosenström P.
        Nucleic Acids Res. 2010; 38: W545-W549
        • Benach-Andreu J.
        X-ray Structure Analysis of Short-chain Dehydrogenases/reductases. Karolinska Medico-Kirurgiska Institute, Sweden, Stockholm1999
        • Smilda T.
        • Reinders P.
        • Beintema J.J.
        Biochem. Genet. 1998; 36: 37-49
        • Paithankar K.S.
        • Feller C.
        • Kuettner E.B.
        • Keim A.
        • Grunow M.
        • Sträter N.
        FEBS J. 2007; 274: 5767-5779
        • Yamazawa R.
        • Nakajima Y.
        • Mushiake K.
        • Yoshimoto T.
        • Ito K.
        J. Biochem. 2011; 149: 701-712
        • Thompson J.D.
        • Gibson T.J.
        • Plewniak F.
        • Jeanmougin F.
        • Higgins D.G.
        Nucleic Acids Res. 1997; 25: 4876-4882