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X-ray Structure and Mutational Analysis of the Atrazine Chlorohydrolase TrzN*

  • Jennifer L. Seffernick
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 55108

    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108

    Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Erik Reynolds
    Affiliations
    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Alexander A. Fedorov
    Affiliations
    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Elena Fedorov
    Affiliations
    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Steven C. Almo
    Affiliations
    Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461
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  • Michael J. Sadowsky
    Affiliations
    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108

    Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, Minnesota 55108

    Department of Soil, Water, and Climate, University of Minnesota, St. Paul, Minnesota 55108
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  • Lawrence P. Wackett
    Correspondence
    To whom correspondence should be addressed: Lawrence P. Wackett, Dept. of Biochemistry, Molecular Biology & Biophysics, 140 Gortner Laboratory, 1479 Gortner Ave., University of Minnesota, St. Paul, MN 55108. Tel.: 612-625-3785; Fax: 612-624-5780
    Affiliations
    Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, St. Paul, Minnesota 55108

    BioTechnology Institute, University of Minnesota, St. Paul, Minnesota 55108

    Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, Minnesota 55108
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  • Author Footnotes
    * This work was supported by a grant from Sygenta Crop Protection (to L. P. W. and M. J. S.).
Open AccessPublished:July 20, 2010DOI:https://doi.org/10.1074/jbc.M110.138677
      Atrazine chlorohydrolase, TrzN (triazine hydrolase or atrazine chlorohydrolase 2), initiates bacterial metabolism of the herbicide atrazine by hydrolytic displacement of a chlorine substituent from the s-triazine ring. The present study describes crystal structures and reactivity of wild-type and active site mutant TrzN enzymes. The homodimer native enzyme structure, solved to 1.40 Å resolution, is a (βα)8 barrel, characteristic of members of the amidohydrolase superfamily. TrzN uniquely positions threonine 325 in place of a conserved aspartate that ligates the metal in most mononuclear amidohydrolases superfamily members. The threonine side chain oxygen atom is 3.3 Å from the zinc atom and 2.6 Å from the oxygen atom of zinc-coordinated water. Mutation of the threonine to a serine resulted in a 12-fold decrease in kcat/Km, largely due to kcat, whereas the T325D and T325E mutants had immeasurable activity. The structure and kinetics of TrzN are reminiscent of carbonic anhydrase, which uses a threonine to assist in positioning water for reaction with carbon dioxide. An isosteric substitution in the active site glutamate, E241Q, showed a large diminution in activity with ametryn, no detectable activity with atratone, and a 10-fold decrease with atrazine, when compared with wild-type TrzN. Activity with the E241Q mutant was nearly constant from pH 6.0 to 10.0, consistent with the loss of a proton-donating group. Structures for TrzN-E241Q were solved with bound ametryn and atratone to 1.93 and 1.64 Å resolution, respectively. Both structure and kinetic determinations suggest that the Glu241 side chain provides a proton to N-1 of the s-triazine substrate to facilitate nucleophilic displacement at the adjacent C-2.

      Introduction

      There is substantial evidence that microbes rapidly evolve new enzymes to metabolize anthropogenic chemicals (
      • Seffernick J.L.
      • Wackett L.P.
      ,
      • Copley S.D.
      ). The s-triazine herbicides, such as atrazine, were first introduced into the environment 50 years ago and >2 billion pounds have been applied globally. s-Triazine compounds were initially found to be poorly biodegradable, but more rapid biodegradation is observed today (
      • Krutz L.J.
      • Burke I.C.
      • Reddy K.N.
      • Zablotowicz R.M.
      • Price A.J.
      ). Many atrazine-degrading bacteria have now been isolated (
      • Topp E.
      • Mulbry W.M.
      • Zhu H.
      • Nour S.M.
      • Cuppels D.
      ,
      • Strong L.C.
      • Rosendahl C.
      • Johnson G.
      • Sadowsky M.J.
      • Wackett L.P.
      ,
      • Piutti S.
      • Semon E.
      • Landry D.
      • Hartmann A.
      • Dousset S.
      • Lichtfouse E.
      • Topp E.
      • Soulas G.
      • Martin-Laurent F.
      ). They invariably contain highly conserved genes encoding enzymes that hydrolytically displace substituents from the s-triazine ring carbon atoms to generate cyanuric acid (Fig. 1). The genes, trzN, atzA, atzB, and atzC, are found on plasmids and now are distributed globally (
      • de Souza M.L.
      • Seffernick J.
      • Martinez B.
      • Sadowsky M.J.
      • Wackett L.P.
      ,
      • de Souza M.L.
      • Wackett L.P.
      • Sadowsky M.J.
      ). These observations are consistent with the idea that a new metabolic pathway for atrazine catabolism may have evolved and spread in recent evolutionary times (
      • Shapir N.
      • Mongodin E.F.
      • Sadowsky M.J.
      • Daugherty S.C.
      • Nelson K.E.
      • Wackett L.P.
      ).
      Figure thumbnail gr1
      FIGURE 1Metabolic pathway of atrazine, ametryn, and atratone in bacteria initiated by atrazine chlorohydrolase, TrzN.
      The s-triazine herbicides, such as atrazine (2-chloro-4-isopropylamino-6-ethylamino-1,3,5-triazine) and ametryn (2-thiomethyl-4-isopropylamino-6-ethylamino-1,3,5-triazine), are metabolized readily by dedicated enzymes. The enzymes have been purified to homogeneity and shown to be inactive with structurally analogous pyrimidines and other closely related compounds (
      • Shapir N.
      • Rosendahl C.
      • Johnson G.
      • Andreina M.
      • Sadowsky M.J.
      • Wackett L.P.
      ,
      • Shapir N.
      • Pedersen C.
      • Gil O.
      • Strong L.
      • Seffernick J.
      • Sadowsky M.J.
      • Wackett L.P.
      ,
      • de Souza M.L.
      • Sadowsky M.J.
      • Wackett L.P.
      ,
      • Seffernick J.L.
      • McTavish H.
      • Osborne J.P.
      • de Souza M.L.
      • Sadowsky M.J.
      • Wackett L.P.
      ). Moreover, the enzymes are isolated from bacteria obtained from herbicide-contaminated environments. Bacteria that completely assimilate atrazine initiate metabolism via an initial dechlorination reaction catalyzed by atrazine chlorohydrolase. Atrazine chlorohydrolase has been used for herbicide remediation and shown to reduce atrazine levels by 90% in a contaminated soil (
      • Strong L.C.
      • McTavish H.
      • Sadowsky M.J.
      • Wackett L.P.
      ). Two distinct atrazine chlorohydrolases, TrzN (
      • Topp E.
      • Mulbry W.M.
      • Zhu H.
      • Nour S.M.
      • Cuppels D.
      ,
      • Shapir N.
      • Pedersen C.
      • Gil O.
      • Strong L.
      • Seffernick J.
      • Sadowsky M.J.
      • Wackett L.P.
      ) and AtzA (atrazine chlorohydrolase 1) (
      • Seffernick J.L.
      • McTavish H.
      • Osborne J.P.
      • de Souza M.L.
      • Sadowsky M.J.
      • Wackett L.P.
      ), have been described. Though both enzymes likely evolved recently, their pairwise protein sequence identity of 28% suggests that they each evolved independently from two different but evolutionarily related ancestors (
      • Shapir N.
      • Pedersen C.
      • Gil O.
      • Strong L.
      • Seffernick J.
      • Sadowsky M.J.
      • Wackett L.P.
      ). However, both TrzN and AtzA catalyze hydrolytic dechlorination reactions with atrazine and related s-triazine herbicides.
      X-ray structures have been determined for chlorohydrolases that act on haloaliphatic and halobenzene substrates. Haloalkane dehalogenase, haloacid dehalogenase, and 4-chlorobenzoyl-CoA dehalogenase all use an aspartate nucleophile and hydrolyze the intermediate enzyme-substrate ester to catalyze an overall hydrolytic displacement reaction (
      • De Jong R.M.
      • Dijkstra B.W.
      ). cis-3-Chloroacrylic acid dehalogenase proceeds via a hydratase-like mechanism (
      • de Jong R.M.
      • Bazzacco P.
      • Poelarends G.J.
      • Johnson Jr., W.H.
      • Kim Y.J.
      • Burks E.A.
      • Serrano H.
      • Thunnissen A.M.
      • Whitman C.P.
      • Dijkstra B.W.
      ). These structurally defined dehalogenases, which use water as a cosubstrate, are not metalloenzymes. In contrast, TrzN and AtzA each require a divalent metal ion to catalyze hydrolytic dehalogenation and thus differ mechanistically from other well studied halidohydrolases (
      • Topp E.
      • Mulbry W.M.
      • Zhu H.
      • Nour S.M.
      • Cuppels D.
      ,
      • Shapir N.
      • Pedersen C.
      • Gil O.
      • Strong L.
      • Seffernick J.
      • Sadowsky M.J.
      • Wackett L.P.
      ,
      • Seffernick J.L.
      • McTavish H.
      • Osborne J.P.
      • de Souza M.L.
      • Sadowsky M.J.
      • Wackett L.P.
      ).
      TrzN and AtzA are both metalloenzymes of the amidohydrolase superfamily that contain zinc(II) and iron(II), respectively (
      • Shapir N.
      • Pedersen C.
      • Gil O.
      • Strong L.
      • Seffernick J.
      • Sadowsky M.J.
      • Wackett L.P.
      ,
      • Seffernick J.L.
      • McTavish H.
      • Osborne J.P.
      • de Souza M.L.
      • Sadowsky M.J.
      • Wackett L.P.
      ). A number of well studied amidohydrolases, such as adenosine deaminase (
      • Wilson D.K.
      • Rudolph F.B.
      • Quiocho F.A.
      ) and cytosine deaminase (
      • Ireton G.C.
      • McDermott G.
      • Black M.E.
      • Stoddard B.L.
      ), catalyze hydrolytic deamination from diazine ring systems, which is somewhat comparable to hydrolytic dechlorination from an s-triazine ring substrate. Yet, AtzA has a high degree of substrate specificity, catalyzing only dehalogenation reactions (
      • Shapir N.
      • Rosendahl C.
      • Johnson G.
      • Andreina M.
      • Sadowsky M.J.
      • Wackett L.P.
      ). In contrast, TrzN has a broader substrate specificity and displaces thiomethyl and methoxy groups from the related s-triazine herbicides ametryn and atratone (2-methoxy-4-isopropylamino-6-ethylamino-1,3,5-triazine), respectively. TrzN has been demonstrated to be present in atrazine-degrading bacteria isolated from four different continents (
      • Shapir N.
      • Mongodin E.F.
      • Sadowsky M.J.
      • Daugherty S.C.
      • Nelson K.E.
      • Wackett L.P.
      ). TrzN has a higher kcat/Km for s-triazine herbicides than does AtzA, making it preferable for remediation of s-triazine herbicide contamination in natural and engineered environments.
      Here, we investigated the structural and catalytic properties of TrzN by analyzing three crystal structures and two sets of active site mutants. The 1.4 Å crystal structure of the wild-type enzyme reveals it to be a unique member of the amidohydrolase superfamily with a four-coordinate metal center and lacking an aspartate ligand present in other proteins in the amidohydrolase superfamily (
      • Seibert C.M.
      • Raushel F.M.
      ). The aspartate in TrzN is replaced by a threonine residue that is within hydrogen-bonding distance to the zinc-bound water. In this regard, TrzN resembles carbonic anhydrase (CA).
      The abbreviations used are: CA
      carbonic anhydrase
      ADDA
      adenosine deaminase
      CYDA
      cytosine deaminase
      GUDA
      guanine deaminase
      MOPS
      4-morpholinepropanesulfonic acid
      CAPS
      3-(cyclohexylamino)propanesulfonic acid.
      Crystal structures also were determined with a catalytically impaired TrzN-E241Q, containing bound ametryn or atratone, respectively. The data suggests that Glu241 in TrzN acts as a proton donor, a function comparable to the conserved glutamate in several other amidohydrolases. Taken together, these data provide insights into enzyme evolution of naturally occurring, substrate-specific enzymes that act to remove anthropogenic chemicals from the environment.

      REFERENCES

        • Seffernick J.L.
        • Wackett L.P.
        Biochemistry. 2001; 40: 12747-12753
        • Copley S.D.
        Nat. Chem. Biol. 2009; 5: 559-566
        • Krutz L.J.
        • Burke I.C.
        • Reddy K.N.
        • Zablotowicz R.M.
        • Price A.J.
        Weed Sci. 2009; 57: 427-434
        • Topp E.
        • Mulbry W.M.
        • Zhu H.
        • Nour S.M.
        • Cuppels D.
        Appl. Environ. Microbiol. 2000; 66: 3134-3141
        • Strong L.C.
        • Rosendahl C.
        • Johnson G.
        • Sadowsky M.J.
        • Wackett L.P.
        Appl. Environ. Microbiol. 2002; 68: 5973-5980
        • Piutti S.
        • Semon E.
        • Landry D.
        • Hartmann A.
        • Dousset S.
        • Lichtfouse E.
        • Topp E.
        • Soulas G.
        • Martin-Laurent F.
        FEMS Microbiol. Lett. 2003; 221: 111-117
        • de Souza M.L.
        • Seffernick J.
        • Martinez B.
        • Sadowsky M.J.
        • Wackett L.P.
        J. Bacteriol. 1998; 180: 1951-1954
        • de Souza M.L.
        • Wackett L.P.
        • Sadowsky M.J.
        Appl. Environ. Microbiol. 1998; 64: 2323-2326
        • Shapir N.
        • Mongodin E.F.
        • Sadowsky M.J.
        • Daugherty S.C.
        • Nelson K.E.
        • Wackett L.P.
        J. Bacteriol. 2007; 189: 674-682
        • Shapir N.
        • Rosendahl C.
        • Johnson G.
        • Andreina M.
        • Sadowsky M.J.
        • Wackett L.P.
        Appl. Environ. Microbiol. 2005; 71: 2214-2220
        • Shapir N.
        • Pedersen C.
        • Gil O.
        • Strong L.
        • Seffernick J.
        • Sadowsky M.J.
        • Wackett L.P.
        J. Bacteriol. 2006; 188: 5859-5864
        • de Souza M.L.
        • Sadowsky M.J.
        • Wackett L.P.
        J. Bacteriol. 1996; 178: 4894-4900
        • Seffernick J.L.
        • McTavish H.
        • Osborne J.P.
        • de Souza M.L.
        • Sadowsky M.J.
        • Wackett L.P.
        Biochemistry. 2002; 41: 14430-14437
        • Strong L.C.
        • McTavish H.
        • Sadowsky M.J.
        • Wackett L.P.
        Environ. Microbiol. 2000; 2: 91-98
        • De Jong R.M.
        • Dijkstra B.W.
        Curr. Op. Struct. Biol. 2003; 13: 722-730
        • de Jong R.M.
        • Bazzacco P.
        • Poelarends G.J.
        • Johnson Jr., W.H.
        • Kim Y.J.
        • Burks E.A.
        • Serrano H.
        • Thunnissen A.M.
        • Whitman C.P.
        • Dijkstra B.W.
        J. Biol. Chem. 2007; 282: 2440-2449
        • Wilson D.K.
        • Rudolph F.B.
        • Quiocho F.A.
        Science. 1991; 252: 1278-1284
        • Ireton G.C.
        • McDermott G.
        • Black M.E.
        • Stoddard B.L.
        J. Mol. Biol. 2002; 315: 687-697
        • Seffernick J.L.
        • Johnson G.
        • Sadowsky M.J.
        • Wackett L.P.
        Appl. Environ. Microbiol. 2000; 66: 4247-4252
        • Seibert C.M.
        • Raushel F.M.
        Biochemistry. 2005; 44: 6383-6391
        • Huang C.C.
        • Novak W.R.
        • Babbitt P.C.
        • Jewett A.I.
        • Ferrin T.E.
        • Klein T.E.
        Pac. Symp. Biocomput. 2000; 12: 230-241
        • Pegg S.C.
        • Brown S.
        • Ojha S.
        • Seffernick J.
        • Meng E.C.
        • Morris J.H.
        • Chang P.J.
        • Huang C.C.
        • Ferrin T.E.
        • Babbitt P.C.
        Biochemistry. 2006; 45: 2545-2555
        • Morris J.H.
        • Huang C.C.
        • Babbitt P.C.
        • Ferrin T.E.
        Bioinformatics. 2007; 23: 2345-2347
        • Seffernick J.L.
        • Samanta S.K.
        • Louie T.M.
        • Wackett L.P.
        • Subramanian M.
        J Biotechnol. 2009; 143: 17-26
        • Otwinowski Z.
        • Minor W.
        Carter C.W.J. Sweet R.M. Abelson J.N. Simon M.I. Methods in Enzymology. Academic Pres, New York1997: 307-326
        • Long F.
        • Vagin A.
        • Young P.
        • Murshudov G.N.
        Acta. Crystallogr. D. 2008; 64: 125-132
        • Emsley P.
        • Cowtan K.
        Acta. Crystallogr. D. 2004; 60: 2126-2132
        • Adams P.D.
        • Afonine P.V.
        • Bunkoczi G.V.B.C.
        • Davis I.W.
        • Echols N.
        • Headd J.J.
        • Hung L.W.
        • Kapral G.J.
        • Grosse-Kunstleve R.W.
        • McCoy A.J.
        • Moriarty N.W.
        • Oeffner R.
        • Read R.J.
        • Richardson J.S.
        • Terwilliger T.C.
        • Zwart P.H.
        Acta Crystallogr. D. 2010; 66: 213-221
        • Lamzin V.S.
        • Wilson K.S.
        Acta. Crystallogr. D. 1993; 49: 129-147
        • Hermann J.C.
        • Marti-Arbona R.
        • Fedorov A.A.
        • Fedorov E.
        • Almo S.C.
        • Shoichet B.K.
        • Raushel F.M.
        Nature. 2007; 448: 775-779
        • Sideraki V.
        • Wilson D.K.
        • Kurz L.C.
        • Quiocho F.A.
        • Rudolph F.B.
        Biochemistry. 1996; 35: 15019-15028
        • Lindskog S.
        Pharmacol. 1997; 74: 1-20
        • Ippolito J.A.
        • Baird T.T.J.
        • McGee S.A.
        • Christianson D.W.
        • Fierke C.A.
        Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 5017-5021
        • Chen X.
        • Tu C.
        • LoGrasso P.V.
        • Laipis P.J.
        • Silverman D.N.
        Biochemistry. 1993; 32: 7861-7865
        • Krebs J.F.
        • Ippolito J.A.
        • Christianson D.W.
        • Fierke C.A.
        J. Biol. Chem. 1993; 268: 27458-27466
        • Huang S.
        • Sjöblom B.
        • Sauer-Eriksson A.E.
        • Jonsson B.H.
        Biochemistry. 2002; 41: 7628-7635
        • Bhaumik D.
        • Medina J.
        • Gathy K.
        • Coleman M.S.
        J. Biol. Chem. 1993; 268: 5464-5470
        • Mohamedali K.A.
        • Kurz L.C.
        • Rudolph F.B.
        Biochemistry. 1996; 35: 1672-1680
        • Meyer A.H.
        • Penning H.
        • Elsner M.
        Environ. Sci. Technol. 2009; 43: 8079-8085
        • Scott C.
        • Jackson C.J.
        • Coppin C.W.
        • Mourant R.G.
        • Hilton M.E.
        • Sutherland T.D.
        • Russell R.J.
        • Oakeshott J.G.
        Appl. Environ Microbiol. 2009; 75: 2184-2191