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Retuning Rieske-type Oxygenases to Expand Substrate Range*

Open AccessPublished:June 08, 2011DOI:https://doi.org/10.1074/jbc.M111.255174
      Rieske-type oxygenases are promising biocatalysts for the destruction of persistent pollutants or for the synthesis of fine chemicals. In this work, we explored pathways through which Rieske-type oxygenases evolve to expand their substrate range. BphAEp4, a variant biphenyl dioxygenase generated from Burkholderia xenovorans LB400 BphAELB400 by the double substitution T335A/F336M, and BphAERR41, obtained by changing Asn338, Ile341, and Leu409 of BphAEp4 to Gln338, Val341, and Phe409, metabolize dibenzofuran two and three times faster than BphAELB400, respectively. Steady-state kinetic measurements of single- and multiple-substitution mutants of BphAELB400 showed that the single T335A and the double N338Q/L409F substitutions contribute significantly to enhanced catalytic activity toward dibenzofuran. Analysis of crystal structures showed that the T335A substitution relieves constraints on a segment lining the catalytic cavity, allowing a significant displacement in response to dibenzofuran binding. The combined N338Q/L409F substitutions alter substrate-induced conformational changes of protein groups involved in subunit assembly and in the chemical steps of the reaction. This suggests a responsive induced fit mechanism that retunes the alignment of protein atoms involved in the chemical steps of the reaction. These enzymes can thus expand their substrate range through mutations that alter the constraints or plasticity of the catalytic cavity to accommodate new substrates or that alter the induced fit mechanism required to achieve proper alignment of reaction-critical atoms or groups.

      Introduction

      Rieske-type oxygenases (ROs)
      The abbreviations used are: RO
      Rieske-type oxygenase
      IPTG
      isopropyl β-d-thiogalactopyranoside
      MES
      4-morpholineethanesulfonic acid.
      catalyze a stereospecific oxygenation of many aromatic and hetero-aromatic molecules. These enzymes have potential applications as biocatalysts to degrade persistent pollutants, such as polyaromatic hydrocarbons (
      • Jakoncic J.
      • Jouanneau Y.
      • Meyer C.
      • Stojanoff V.
      ,
      • Kauppi B.
      • Lee K.
      • Carredano E.
      • Parales R.E.
      • Gibson D.T.
      • Eklund H.
      • Ramaswamy S.
      ), polychlorinated biphenyls (
      • Furukawa K.
      • Suenaga H.
      • Goto M.
      ,
      • Haddock J.D.
      • Horton J.R.
      • Gibson D.T.
      ,
      • Sylvestre M.
      ), and chlorodibenzofurans (
      • Chang Y.S.
      ,
      • Mohammadi M.
      • Sylvestre M.
      ), or to produce chiral arene cis-dihydrodiols of interest in enantioselective syntheses for the manufacture of fine chemicals (
      • Chun H.K.
      • Ohnishi Y.
      • Shindo K.
      • Misawa N.
      • Furukawa K.
      • Horinouchi S.
      ,
      • Seeger M.
      • González M.
      • Cámara B.
      • Muñoz L.
      • Ponce E.
      • Mejías L.
      • Mascayano C.
      • Vásquez Y.
      • Sepúlveda-Boza S.
      ,
      • Seo J.
      • Kang S.I.
      • Won D.
      • Kim M.
      • Ryu J.Y.
      • Kang S.W.
      • Um B.H.
      • Pan C.H.
      • Ahn J.H.
      • Chong Y.
      • Kanaly R.A.
      • Han J.
      • Hur H.G.
      ). Biphenyl dioxygenase, one of the most extensively studied ROs, catalyzes the first reaction of the bacterial biphenyl catabolic pathway. Biphenyl dioxygenase has three components: the iron-sulfur oxygenase (hereinafter referred to as BphAE), a heterohexamer comprised of three α (Mr = 51,000) and three β (Mr = 22,000) subunits; the ferredoxin (BphF, Mr = 12,000); and the ferredoxin reductase (BphG, Mr = 43,000). The encoding genes for Burkholderia xenovorans LB400 (
      • Erickson B.D.
      • Mondello F.J.
      ), which is the best polychlorinated biphenyl degrader of natural origin, are bphA (BphAELB400 α subunit), bphE (BphAELB400 β subunit), bphF (BphFLB400), and bphG (BphGLB400) (see Fig. 1).
      Figure thumbnail gr1
      FIGURE 1Biphenyl dioxygenase reaction. The inset shows the structure of dibenzofuran and possible positions of oxygenation. A, angular; L, lateral.
      BphAELB400 has been thoroughly investigated because it can oxygenate a broad range of substrates. BphAELB400 variants with extended substrate range have been generated by site-directed mutagenesis (
      • Mondello F.J.
      • Turcich M.P.
      • Lobos J.H.
      • Erickson B.D.
      ) and directed evolution (
      • Barriault D.
      • Plante M.M.
      • Sylvestre M.
      ,
      • Barriault D.
      • Sylvestre M.
      ). However, the enzyme's structural features that modulate substrate range and catalytic efficiency have yet to be determined. Many investigations have identified residues in contact with the substrate, or removed from it, as key determinants of substrate preference and regiospecificity (
      • Mondello F.J.
      • Turcich M.P.
      • Lobos J.H.
      • Erickson B.D.
      ,
      • Barriault D.
      • Plante M.M.
      • Sylvestre M.
      ,
      • Barriault D.
      • Sylvestre M.
      ,
      • Ferraro D.J.
      • Okerlund A.L.
      • Mowers J.C.
      • Ramaswamy S.
      ,
      • Furusawa Y.
      • Nagarajan V.
      • Tanokura M.
      • Masai E.
      • Fukuda M.
      • Senda T.
      ,
      • Kumar P.
      • Mohammadi M.
      • Viger J.F.
      • Barriault D.
      • Gomez-Gil L.
      • Eltis L.D.
      • Bolin J.T.
      • Sylvestre M.
      ,
      • Suenaga H.
      • Goto M.
      • Furukawa K.
      ,
      • Suenaga H.
      • Mitsuoka M.
      • Ura Y.
      • Watanabe T.
      • Furukawa K.
      ,
      • Suenaga H.
      • Nishi A.
      • Watanabe T.
      • Sakai M.
      • Furukawa K.
      ,
      • Vézina J.
      • Barriault D.
      • Sylvestre M.
      ,
      • Zielinski M.
      • Kahl S.
      • Hecht H.J.
      • Hofer B.
      ,
      • Zielinski M.
      • Kahl S.
      • Standfuss-Gabisch C.
      • Cámara B.
      • Seeger M.
      • Hofer B.
      ). Recently Thr335 of BphAELB400, which is removed from the substrate, was found to restrain the range of chlorobiphenyls the enzyme can oxidize by controlling the spatial distribution of protein atoms in contact with the substrates (
      • Kumar P.
      • Mohammadi M.
      • Viger J.F.
      • Barriault D.
      • Gomez-Gil L.
      • Eltis L.D.
      • Bolin J.T.
      • Sylvestre M.
      ). Changing Thr335 to Ala relieves intramolecular constraints on Gly321, allowing for significant movement of this residue during substrate binding, thereby increasing the space available to accommodate the bulkier substrate 2,6-dichlorobiphenyl. In addition, crystal structures of the oxygenase component of carbazole 1,9a-dioxygenase and of the binary complex of the oxygenase and ferredoxin components provided evidence that conformational changes are required to suitably align the Rieske clusters of the ferredoxin and oxygenase components (
      • Inoue K.
      • Ashikawa Y.
      • Umeda T.
      • Abo M.
      • Katsuki J.
      • Usami Y.
      • Noguchi H.
      • Fujimoto Z.
      • Terada T.
      • Yamane H.
      • Nojiri H.
      ). These observations are consistent with a mechanism whereby an induced fit process is involved in RO substrate binding and catalytic function. Understanding how conformational adjustments influence the turnover rate and how they can be modified to enhance activity toward new substrates will aid the development of new, better performing catalysts.
      Unlike biphenyl, the fused rings of dibenzofuran are locked in a co-planar conformation (Fig. 1), and this molecule is poorly oxygenated by BphAELB400 (
      • L'Abbée J.B.
      • Barriault D.
      • Sylvestre M.
      ,
      • Seeger M.
      • Cámara B.
      • Hofer B.
      ). In previous reports (
      • Mohammadi M.
      • Sylvestre M.
      ,
      • Barriault D.
      • Sylvestre M.
      ), we described variant BphAEp4 obtained by the double substitution of Thr335-Phe336 of BphAELB400 to Ala335-Met336 and variant BphAERR41 obtained by changing Asn338, Ile341, and Leu409 of BphAp4 to Gln338, Val341, and Phe409. BphAERR41 was selected by directed evolution for its higher turnover rate with dibenzofuran than the parent BphAEp4 (
      • Mohammadi M.
      • Sylvestre M.
      ). The library was produced by changing Asn338 and Ile341 of BphAEp4 simultaneously by saturation mutagenesis (
      • Mohammadi M.
      • Sylvestre M.
      ), but an additional spontaneous mutation L409F occurred in this mutant.
      In this study, to gain more insight into the pathways through which ROs evolve to expand their substrate range, we identified the mutations in BphAEp4 and in BphAERR41 that contribute most to their enhanced activity toward dibenzofuran and analyzed the crystal structures of BphAERR41 and its dibenzofuran-bound form to evaluate the consequences of the mutations.

      DISCUSSION

      In this study, we examined the crystal structure of BphAERR41, an evolved RO that oxidizes dibenzofuran more efficiently than its BphAELB400 and BphAEp4 parents. Despite the limitations of crystal structure analyses, the study revealed two pathways through which ROs evolve to expand their substrate range.
      Traditionally, enzyme engineering to alter the substrate range involves mutations at residues lining the catalytic pocket. This approach has been applied successfully in many circumstances (
      • Bak-Jensen K.S.
      • André G.
      • Gottschalk T.E.
      • Paës G.
      • Tran V.
      • Svensson B.
      ,
      • Dietrich M.
      • Do T.A.
      • Schmid R.D.
      • Pleiss J.
      • Urlacher V.B.
      ,
      • Huang W.C.
      • Westlake A.C.
      • Maréchal J.D.
      • Joyce M.G.
      • Moody P.C.
      • Roberts G.C.
      ,
      • Ostrander E.L.
      • Larson J.D.
      • Schuermann J.P.
      • Tanner J.J.
      ,
      • Parales R.E.
      • Lee K.
      • Resnick S.M.
      • Jiang H.
      • Lessner D.J.
      • Gibson D.T.
      ,
      • Suenaga H.
      • Goto M.
      • Furukawa K.
      ,
      • Xu D.
      • Enroth C.
      • Lindqvist Y.
      • Ballou D.P.
      • Massey V.
      ). Reducing the size of a side chain or altering charge distributions can generate enzyme with new catalytic properties.
      However, other studies have shown that several residues not in direct contact with the substrate can significantly change the catalytic properties of biphenyl dioxygenase toward biphenyl analogs (
      • Vézina J.
      • Barriault D.
      • Sylvestre M.
      ,
      • Zielinski M.
      • Kahl S.
      • Hecht H.J.
      • Hofer B.
      ,
      • Suenaga H.
      • Goto M.
      • Furukawa K.
      ). In this work we confirm the importance of the T335A mutation. In altering the plasticity of the catalytic cavity, this mutation allows the carbonyl of residue Gly321 to move away from the substrate. In a previous work, we showed that this movement was required to increase the space available to bind the bulky 2,6-dichlorobiphenyl in a productive orientation (
      • Kumar P.
      • Mohammadi M.
      • Viger J.F.
      • Barriault D.
      • Gomez-Gil L.
      • Eltis L.D.
      • Bolin J.T.
      • Sylvestre M.
      ). Because dibenzofuran is obligatory co-planar, any misplacement of the distal ring would influence the orientation of the proximal ring inside the catalytic pocket. Therefore, consistent with an induced fit mechanism, in BphAEp4 and BphAERR41, the displacement of Gly321 appears to be required to reduce the influence it exerts through atomic interactions on the substrate's distal ring.
      In this work, we highlighted a second and more subtle route to changes in substrate range, which implies that in ROs, either one or both of the induced fit or protein dynamic processes are involved to place the protein atoms involved in the reaction into proper relationships that facilitate catalysis. The reaction catalyzed by ROs is complex; it not only involves substrate binding and release of product, but also one dioxygen molecule is required in the reaction, and electrons must be transferred from the ferredoxin component to the Rieske cluster of one α subunit and then to the catalytic iron of the vicinal α subunit. Furthermore, a recent report showed residues at the interface between the Rieske domain and the catalytic domain move during formation of the complex between the oxygenase and ferredoxin components of carbazole 1,9a-dioxygenase (
      • Inoue K.
      • Ashikawa Y.
      • Umeda T.
      • Abo M.
      • Katsuki J.
      • Usami Y.
      • Noguchi H.
      • Fujimoto Z.
      • Terada T.
      • Yamane H.
      • Nojiri H.
      ). This implies that reaction-critical atoms from the Rieske domain must align properly with those of the vicinal catalytic domain, and the reaction-critical atoms of the catalytic domain must align properly to work together during the catalytic process. Structural analysis shows that residues located on secondary structures α6 and α12 are involved in subunit assembly, and biochemical data suggest that they are involved in the catalytic reaction (electron transfer and protonation) (
      • Kauppi B.
      • Lee K.
      • Carredano E.
      • Parales R.E.
      • Gibson D.T.
      • Eklund H.
      • Ramaswamy S.
      ,
      • Parales R.E.
      • Parales J.V.
      • Gibson D.T.
      ). The fact that these residues move during substrate binding is consistent with a substrate-induced retuning process required to suitably align the protein atoms involved in the chemical steps of the reaction. In such a context, by altering the interactions occurring between secondary structure elements surrounding the catalytic center, the N338Q mutation generates a protein unable to stabilize the α3β3 assembly previously shown to be required for activity (
      • Hurtubise Y.
      • Barriault D.
      • Sylvestre M.
      ). However, the double N338Q and L409F substitution generates an α subunit that supports a stable hexamer and where the retuning process is improved compared with its BphAELB400 and BphAEp4 parents, resulting in a more efficient and faster catalytic reaction. ROs can thus be engineered to enhance their catalytic properties toward new substrates by altering the process involved in fine-tuning the interplay between the reaction-critical atoms.
      Many questions remain unanswered; crystal structure analysis did not determine a clear-cut mechanism by which the double N338Q/L409F substitution affects the enzyme structure and catalytic properties, and our data do not determine which of the enzymatic steps are accelerated during the reaction. Although the data do not provide any direct demonstration that the N338Q and L409F substitutions either affect an induced fit or protein dynamic mechanism involved in the catalytic reaction, it is clear from crystal structure analysis that these residues occupy strategic positions whereby they can interact with reaction-critical protein atoms/groups and affect oligomeric assembly. Furthermore, residues of helix α12 and α6, and especially Asp388 and Gln226, which are postulated to play a key role in the catalytic reaction (
      • Karlsson A.
      • Parales J.V.
      • Parales R.E.
      • Gibson D.T.
      • Eklund H.
      • Ramaswamy S.
      ), moved significantly during substrate binding in all variants (BphAELB400, BphAEp4, and BphAERR41).
      Altogether, our analysis shows that evolving ROs to change their substrate specificity is a rather complex enterprise that does not exclusively involve mutations at key residues in direct contact with the substrate. It appears that some mutations affect key residues associated with necessary conformational changes that are more difficult to identify by a rational approach but that are required to allow productive or improved interplay of reaction-critical atoms both inside and outside the substrate-binding pocket.

      Acknowledgments

      We appreciate the use of the SE Regional Collaborative Access Team 22-ID Beamline in the collection of x-ray diffraction data.

      REFERENCES

        • Jakoncic J.
        • Jouanneau Y.
        • Meyer C.
        • Stojanoff V.
        Biochem. Biophys. Res. Commun. 2007; 352: 861-866
        • Kauppi B.
        • Lee K.
        • Carredano E.
        • Parales R.E.
        • Gibson D.T.
        • Eklund H.
        • Ramaswamy S.
        Structure. 1998; 6: 571-586
        • Furukawa K.
        • Suenaga H.
        • Goto M.
        J. Bacteriol. 2004; 186: 5189-5196
        • Haddock J.D.
        • Horton J.R.
        • Gibson D.T.
        J. Bacteriol. 1995; 177: 20-26
        • Sylvestre M.
        Int. Biodeter. Biodegr. 2004; 54: 153-162
        • Chang Y.S.
        J. Mol. Microbiol. Biotechnol. 2008; 15: 152-171
        • Mohammadi M.
        • Sylvestre M.
        Chem. Biol. 2005; 12: 835-846
        • Chun H.K.
        • Ohnishi Y.
        • Shindo K.
        • Misawa N.
        • Furukawa K.
        • Horinouchi S.
        J. Mol. Catal. B Enzym. 2003; 21: 113-121
        • Seeger M.
        • González M.
        • Cámara B.
        • Muñoz L.
        • Ponce E.
        • Mejías L.
        • Mascayano C.
        • Vásquez Y.
        • Sepúlveda-Boza S.
        Appl. Environ. Microbiol. 2003; 69: 5045-5050
        • Seo J.
        • Kang S.I.
        • Won D.
        • Kim M.
        • Ryu J.Y.
        • Kang S.W.
        • Um B.H.
        • Pan C.H.
        • Ahn J.H.
        • Chong Y.
        • Kanaly R.A.
        • Han J.
        • Hur H.G.
        Appl. Microbiol. Biotechnol. 2011; 89: 1773-1782
        • Erickson B.D.
        • Mondello F.J.
        J. Bacteriol. 1992; 174: 2903-2912
        • Mondello F.J.
        • Turcich M.P.
        • Lobos J.H.
        • Erickson B.D.
        Appl. Environ. Microbiol. 1997; 63: 3096-3103
        • Barriault D.
        • Plante M.M.
        • Sylvestre M.
        J. Bacteriol. 2002; 184: 3794-3800
        • Barriault D.
        • Sylvestre M.
        J. Biol. Chem. 2004; 279: 47480-47488
        • Ferraro D.J.
        • Okerlund A.L.
        • Mowers J.C.
        • Ramaswamy S.
        J. Bacteriol. 2006; 188: 6986-6994
        • Furusawa Y.
        • Nagarajan V.
        • Tanokura M.
        • Masai E.
        • Fukuda M.
        • Senda T.
        J. Mol. Biol. 2004; 342: 1041-1052
        • Kumar P.
        • Mohammadi M.
        • Viger J.F.
        • Barriault D.
        • Gomez-Gil L.
        • Eltis L.D.
        • Bolin J.T.
        • Sylvestre M.
        J. Mol. Biol. 2011; 405: 531-547
        • Suenaga H.
        • Goto M.
        • Furukawa K.
        J. Biol. Chem. 2001; 276: 22500-22506
        • Suenaga H.
        • Mitsuoka M.
        • Ura Y.
        • Watanabe T.
        • Furukawa K.
        J. Bacteriol. 2001; 183: 5441-5444
        • Suenaga H.
        • Nishi A.
        • Watanabe T.
        • Sakai M.
        • Furukawa K.
        J. Biosci. Bioeng. 1999; 87: 430-435
        • Vézina J.
        • Barriault D.
        • Sylvestre M.
        J. Bacteriol. 2007; 189: 779-788
        • Zielinski M.
        • Kahl S.
        • Hecht H.J.
        • Hofer B.
        J. Bacteriol. 2003; 185: 6976-6980
        • Zielinski M.
        • Kahl S.
        • Standfuss-Gabisch C.
        • Cámara B.
        • Seeger M.
        • Hofer B.
        Appl. Environ. Microbiol. 2006; 72: 2191-2199
        • Inoue K.
        • Ashikawa Y.
        • Umeda T.
        • Abo M.
        • Katsuki J.
        • Usami Y.
        • Noguchi H.
        • Fujimoto Z.
        • Terada T.
        • Yamane H.
        • Nojiri H.
        J. Mol. Biol. 2009; 392: 436-451
        • L'Abbée J.B.
        • Barriault D.
        • Sylvestre M.
        Appl. Microbiol. Biotechnol. 2005; 67: 506-514
        • Seeger M.
        • Cámara B.
        • Hofer B.
        J. Bacteriol. 2001; 183: 3548-3555
        • Lin J.J.
        • Smith M.
        • Jessee J.
        • Bloom F.
        BioTechniques. 1992; 12: 718-721
        • Miroux B.
        • Walker J.E.
        J. Mol. Biol. 1996; 260: 289-298
        • Sambrook J.
        • Fritsch E.F.
        • Maniatis T.
        Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989
        • Laemmli U.K.
        Nature. 1970; 227: 680-685
        • Hurtubise Y.
        • Barriault D.
        • Sylvestre M.
        J. Biol. Chem. 1996; 271: 8152-8156
        • Haddock J.D.
        • Gibson D.T.
        J. Bacteriol. 1995; 177: 5834-5839
        • Hurtubise Y.
        • Barriault D.
        • Powlowski J.
        • Sylvestre M.
        J. Bacteriol. 1995; 177: 6610-6618
        • Imbeault N.Y.
        • Powlowski J.B.
        • Colbert C.L.
        • Bolin J.T.
        • Eltis L.D.
        J. Biol. Chem. 2000; 275: 12430-12437
        • Kumar P.
        • Gómez-Gil L.
        • Mohammadi M.
        • Sylvestre M.
        • Eltis L.D.
        • Bolin J.T.
        Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2011; 67: 59-62
        • Karlsson A.
        • Parales J.V.
        • Parales R.E.
        • Gibson D.T.
        • Eklund H.
        • Ramaswamy S.
        Science. 2003; 299: 1039-1042
        • Dundas J.
        • Ouyang Z.
        • Tseng J.
        • Binkowski A.
        • Turpaz Y.
        • Liang J.
        Nucleic Acids Res. 2006; 34: W116-W118
        • Parales R.E.
        • Parales J.V.
        • Gibson D.T.
        J. Bacteriol. 1999; 181: 1831-1837
        • Bak-Jensen K.S.
        • André G.
        • Gottschalk T.E.
        • Paës G.
        • Tran V.
        • Svensson B.
        J. Biol. Chem. 2004; 279: 10093-10102
        • Dietrich M.
        • Do T.A.
        • Schmid R.D.
        • Pleiss J.
        • Urlacher V.B.
        J. Biotechnol. 2009; 139: 115-117
        • Huang W.C.
        • Westlake A.C.
        • Maréchal J.D.
        • Joyce M.G.
        • Moody P.C.
        • Roberts G.C.
        J. Mol. Biol. 2007; 373: 633-651
        • Ostrander E.L.
        • Larson J.D.
        • Schuermann J.P.
        • Tanner J.J.
        Biochemistry. 2009; 48: 951-959
        • Parales R.E.
        • Lee K.
        • Resnick S.M.
        • Jiang H.
        • Lessner D.J.
        • Gibson D.T.
        J. Bacteriol. 2000; 182: 1641-1649
        • Suenaga H.
        • Goto M.
        • Furukawa K.
        Appl. Microbiol. Biotechnol. 2006; 71: 168-176
        • Xu D.
        • Enroth C.
        • Lindqvist Y.
        • Ballou D.P.
        • Massey V.
        Biochemistry. 2002; 41: 13627-13636