Crystal Structures of the Organomercurial Lyase MerB in Its Free and Mercury-bound Forms

Bacteria resistant to methylmercury utilize two enzymes (MerA and MerB) to degrade methylmercury to the less toxic elemental mercury. The crucial step is the cleavage of the carbon-mercury bond of methylmercury by the organomercurial lyase (MerB). In this study, we determined high resolution crystal structures of MerB in both the free (1.76-Å resolution) and mercury-bound (1.64-Å resolution) states. The crystal structure of free MerB is very similar to the NMR structure, but important differences are observed when comparing the two structures. In the crystal structure, an amino-terminal α-helix that is not present in the NMR structure makes contact with the core region adjacent to the catalytic site. This interaction between the amino-terminal helix and the core serves to bury the active site of MerB. The crystal structures also provide detailed insights into the mechanism of carbon-mercury bond cleavage by MerB. The structures demonstrate that two conserved cysteines (Cys-96 and Cys-159) play a role in substrate binding, carbon-mercury bond cleavage, and controlled product (ionic mercury) release. In addition, the structures establish that an aspartic acid (Asp-99) in the active site plays a crucial role in the proton transfer step required for the cleavage of the carbon-mercury bond. These findings are an important step in understanding the mechanism of carbon-mercury bond cleavage by MerB. Bacteria resistant to methylmercury utilize two enzymes (MerA and MerB) to degrade methylmercury to the less toxic elemental mercury. The crucial step is the cleavage of the carbon-mercury bond of methylmercury by the organomercurial lyase (MerB). In this study, we determined high resolution crystal structures of MerB in both the free (1.76-Å resolution) and mercury-bound (1.64-Å resolution) states. The crystal structure of free MerB is very similar to the NMR structure, but important differences are observed when comparing the two structures. In the crystal structure, an amino-terminal α-helix that is not present in the NMR structure makes contact with the core region adjacent to the catalytic site. This interaction between the amino-terminal helix and the core serves to bury the active site of MerB. The crystal structures also provide detailed insights into the mechanism of carbon-mercury bond cleavage by MerB. The structures demonstrate that two conserved cysteines (Cys-96 and Cys-159) play a role in substrate binding, carbon-mercury bond cleavage, and controlled product (ionic mercury) release. In addition, the structures establish that an aspartic acid (Asp-99) in the active site plays a crucial role in the proton transfer step required for the cleavage of the carbon-mercury bond. These findings are an important step in understanding the mechanism of carbon-mercury bond cleavage by MerB. In the environment, mercury can exist in three different forms: elemental mercury, ionic mercury, and methylmercury. The toxicity of methylmercury is attributed to its lipophilicity and affinity for thiol residues found in proteins (1Clarkson T.W. Magos L. Crit. Rev. Toxicol. 2006; 36: 609-662Crossref PubMed Scopus (1566) Google Scholar). The ability of methylmercury to accumulate in fat tissues leads to its biomagnification within the food chain and ultimately to its toxicity (2Mergler D. Anderson H.A. Chan L.H. Mahaffey K.R. Murray M. Sakamoto M. Stern A.H. Ambio. 2007; 36: 3-11Crossref PubMed Scopus (881) Google Scholar, 3Scheuhammer A.M. Meyer M.W. Sandheinrich M.B. Murray M.W. Ambio. 2007; 36: 12-18Crossref PubMed Scopus (716) Google Scholar). An example of methylmercury bioaccumulation is the high levels found in fish species in contaminated waterways. Select fish species accumulate high levels of methylmercury, and consumption of these contaminated species poses a serious health threat to humans. Because of the high number of mercury-contaminated waterways throughout the world, there is a need to develop an effective remediation system for methylmercury. Most systems for cleanup of mercury-contaminated sediments use either physical or chemical remediation. Unfortunately these systems are expensive and environmentally disruptive. In the 1960s, bacteria were isolated from soils and sediments contaminated with high levels of mercury (4Moore B. Lancet. 1960; 2: 453-458Abstract PubMed Scopus (41) Google Scholar, 5Richmond M.H. John M. Nature. 1964; 202: 1360-1361Crossref PubMed Scopus (53) Google Scholar, 6Novick R.P. Roth C. J. Bacteriol. 1968; 95: 1335-1342Crossref PubMed Google Scholar). Analysis of these bacteria demonstrated that they had acquired a series of plasmid-encoded genes collectively referred to as the mer operon that imparts resistance to mercury. Although the precise composition of the mer operon varies between bacterial strains, strains resistant to high levels of methylmercury code for two enzymes (MerA and MerB) that transform methylmercury to elemental mercury (7Summers A.O. Annu. Rev. Microbiol. 1986; 40: 607-634Crossref PubMed Scopus (250) Google Scholar). The organomercurial lyase MerB cleaves the carbon-mercury bond of methylmercury, releases methane (8Schottel J.L. J. Biol. Chem. 1978; 253: 4341-4349Abstract Full Text PDF PubMed Google Scholar), and directly transfers the ionic mercury to the mercurial reductase MerA. MerA reduces the ionic mercury to elemental mercury, which is volatile and diffuses out of the bacteria (9Summers A.O. Sugarman L.I. J. Bacteriol. 1974; 119: 242-249Crossref PubMed Google Scholar). Because of the unique properties of these two enzymes, there are numerous ongoing attempts to exploit them in bioremediation systems to clean up methylmercury contamination (10Lyyra S. Meagher R.B. Kim T. Heaton A. Montello P. Balish R.S. Merkle S.A. Plant Biotechnol. J. 2007; 5: 254-262Crossref PubMed Scopus (73) Google Scholar, 11Che D. Meagher R.B. Rugh C.L. Kim T. Heaton A.C.P. Merkle S.A. In Vitro Cell Dev. Biol. Plant. 2006; 42: 228-234Crossref Scopus (19) Google Scholar, 12Bizily S.P. Rugh C.L. Meagher R.B. Nat. Biotechnol. 2000; 18: 213-217Crossref PubMed Scopus (265) Google Scholar, 13Bizily S.P. Rugh C.L. Summers A.O. Meagher R.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 6808-6813Crossref PubMed Scopus (196) Google Scholar). MerB cleaves the carbon-mercury bond of many organomercurials to generate ionic mercury and a protonated carbon species (14Begley T.P. Walts A.E. Walsh C.T. Biochemistry. 1986; 25: 7186-7192Crossref PubMed Scopus (63) Google Scholar). Based on kinetic experiments, it was concluded that the reaction mechanism is a bimolecular electrophilic substitution (SE2) (15Begley T.P. Walts A.E. Walsh C.T. Biochemistry. 1986; 25: 7192-7200Crossref PubMed Scopus (89) Google Scholar). In this mechanism, a proton attacks the carbon moiety from the same side as the mercury, and bond cleavage and protonation occur with retention of the stereochemistry at the carbon position. Based on subsequent mutagenesis experiments, more detailed models have been proposed describing the catalytic role of cysteine residues (16Pitts K.E. Summers A.O. Biochemistry. 2002; 41: 10287-10296Crossref PubMed Scopus (65) Google Scholar). Mutagenesis studies with MerB from Escherichia coli (plasmid R831b) demonstrated that two highly conserved cysteines (Cys-96 and Cys-159) were essential for catalytic activity. The NMR structure of free MerB confirmed that these two cysteines are in close proximity within the active site (17Di Lello P. Benison G.C. Valafar H. Pitts K.E. Summers A.O. Legault P. Omichinski J.G. Biochemistry. 2004; 43: 8322-8332Crossref PubMed Scopus (30) Google Scholar). Recently model compounds have been shown to cleave carbon-mercury bonds using only thiol groups, and their unique properties have been proposed to mimic the mechanism of MerB and specifically Cys-96 and Cys-159 (18Melnick J.G. Parkin G. Science. 2007; 317: 225-227Crossref PubMed Scopus (88) Google Scholar). Despite these efforts, the precise details of the mechanism by which MerB cleaves methylmercury are still poorly understood. In this study, we determined high resolution crystal structures of MerB in the free (1.76-Å resolution) and mercury-bound (1.64-Å resolution) states. The two structures of MerB provide important insights into the catalytic mechanism of this unique enzyme. The structures support earlier studies indicating that Cys-96 and Cys-159 are important for the binding to organomercurials and suggest that Asp-99 is an active site residue that participates in the protonolysis of the carbon-mercury bond. Expression of Recombinant Proteins—The sequence encoding MerB from E. coli plasmid R831b was cloned as described previously (17Di Lello P. Benison G.C. Valafar H. Pitts K.E. Summers A.O. Legault P. Omichinski J.G. Biochemistry. 2004; 43: 8322-8332Crossref PubMed Scopus (30) Google Scholar). The MerB mutants (C96S MerB, C159S MerB, and C160S MerB) were prepared by site-directed mutagenesis of plasmid pQZB1 (16Pitts K.E. Summers A.O. Biochemistry. 2002; 41: 10287-10296Crossref PubMed Scopus (65) Google Scholar). Wild-type MerB was expressed and purified as described previously (16Pitts K.E. Summers A.O. Biochemistry. 2002; 41: 10287-10296Crossref PubMed Scopus (65) Google Scholar, 17Di Lello P. Benison G.C. Valafar H. Pitts K.E. Summers A.O. Legault P. Omichinski J.G. Biochemistry. 2004; 43: 8322-8332Crossref PubMed Scopus (30) Google Scholar). Prior to crystallization, proteins were dialyzed (see supplemental methods). Crystallization—Crystals of MerB, C96S MerB, C159S MerB, and C160S MerB were grown by vapor diffusion at 23 °C using either a 1:1 or 1:2 mixture of protein solution (8 mg/ml initial protein concentration) and precipitant buffer, respectively, that was equilibrated against a reservoir of precipitant buffer (see supplemental methods). Data Collection and Processing—MerB crystals were soaked for precise times (7, 10, and 25 min) in organomercurial buffer (precipitant buffer plus a 1.0 mm concentration of either methylmercury or para-hydroxymercury benzoic acid plus 10 mm l-DTT 5The abbreviations used are: DTT, dithiothreitol; [TmBut]K, tris(2-mercapto-1-t-butylimidazoylyl)hydroborate. or cysteine). Diffraction data were collected from single crystals at beam line X29 of the National Synchrotron Light Source (Brookhaven National Laboratory, Upton, NY) using an ADSC Quantum-315 charge-coupled device (Area Detector Systems, Poway, CA), beam line X12b of the National Synchrotron Light Source (Brookhaven National Laboratory) using an ADSC Quantum-4 charge-coupled device (Area Detector Systems), or beam line 22-BM of the Advanced Photon Source (Argonne National Laboratory, Argonne, IL) using an MX-225 system (Rayonix/Mar USA, Evanston, IL). All data sets were processed with HKL2000 (19Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar), and the results are summarized in Table 1 and in supplemental Table 2.TABLE 1Data collection and structural refinement statistics for MerB in the free and mercury-bound formsFree formMercury-bound formSoaking experiment details Organomercurial1 mm PHMBAapara-Hydroxymercury benzoic acid Exogenous thiol10 mm l-Cys Time (min)10Data collection statistics Space groupP21P21 Unit cell parameters a (Å), b (Å), c (Å)38.7, 90.0, 52.238.4, 89.2, 51.6 β (°)100.5100.5 Resolution (Å)20-1.76 (1.82-1.76)bValues in parentheses correspond to the highest resolution shell20-1.64 (1.70-1.64) Unique reflections33,97440,293 Completeness (%)97.9 (90.9)96.6 (93.2) RsymcRsym = Σhkl Σi|Ii(hkl) – Īi(hkl)|/Σhkl ΣiIi(hkl) with i running over the number of independent observations of reflection hkl0.050 (0.181)0.064 (0.481) I/σI25.7 (6.3)17.7 (2.5) Redundancy3.7 (2.8)3.9 (3.5)Model refinement statistics No. of reflections33,44139.084 Rcryst (%)dRcryst = Σhkl||Fo(hkl)| – |Fc(hkl)||/Σhkl|Fo(hkl)|18.218.5 Rfree (%)eRfree = Σhkl∊T||Fo(hkl) – |Fc(hkl)||/Σhkl∊T|Fo(hkl)| where T is a test data set randomly selected from the observed reflections prior to refinement. The test data set was not used throughout refinement20.821.3 Protein atoms3,0843,084 Water molecules347311 Average B-factor (Å2) Protein21.621.9 Waters34.328.7 Estimated coordinate error (Å)fBased on maximum likelihood0.250.18 r.m.s.gRoot mean square deviations Bonds (Å)0.0050.006 Angles (°)0.9741.049 Protein Data Bank code3F0O3F0Pa para-Hydroxymercury benzoic acidb Values in parentheses correspond to the highest resolution shellc Rsym = Σhkl Σi|Ii(hkl) – Īi(hkl)|/Σhkl ΣiIi(hkl) with i running over the number of independent observations of reflection Rcryst = Σhkl||Fo(hkl)| – Rfree = Σhkl∊T||Fo(hkl) – |Fc(hkl)||/Σhkl∊T|Fo(hkl)| where T is a test data set randomly selected from the observed reflections prior to refinement. The test data set was not used throughout Based on maximum mean square in a and used for model of the MerB crystal structure were determined by single plus differences from the mercury using J. Biol. 1999; PubMed Scopus Google Scholar, Biol. 2000; PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). The model was using in with A. Nat. Biol. 1999; PubMed Scopus Google Scholar). The structures were to of refinement and with and NMR P. J. M. T. Biol. PubMed Scopus Google or Biol. 2002; PubMed Scopus Google Scholar, Laboratory, and model using P. Biol. 2004; PubMed Scopus Google Scholar). model with and A. Murray J. 2007; PubMed Scopus Google Scholar), are shown in Table The and structure have been with the Protein Data The were using The Scholar), and the differences between the structures were with PubMed Scopus Google Scholar). and crystals were at and soaked with different organomercurial to generate the mercury-bound MerB. The MerB crystal structure is a in the MerB in solution been shown to (14Begley T.P. Walts A.E. Walsh C.T. Biochemistry. 1986; 25: 7186-7192Crossref PubMed Scopus (63) Google Scholar, 17Di Lello P. Benison G.C. Valafar H. Pitts K.E. Summers A.O. Legault P. Omichinski J.G. Biochemistry. 2004; 43: 8322-8332Crossref PubMed Scopus (30) Google Scholar). between the two and present in the MerB from E. coli is and residues were used to A. residues were used with the of residues and The B-factor for is for the two for and for B. Despite differences in the data the two have between the and NMR of NMR structure demonstrated that MerB of three by that into two an amino-terminal region and a core The crystal structure of free MerB is very similar to the solution structure of the from the amino-terminal region and the core region of the two structures that there are only differences of the of the two structures a number of important The differences are observed in the 25 at the and in a of adjacent to the catalytic site. In addition, the crystal structure a more of of MerB and residues that were not by the NMR In the solution structure, the 25 at the are not of the of In the crystal residues an α-helix that is against the core region of MerB. This results in the amino-terminal region a different to the core region when with the NMR structure In addition, a α-helix been between residues in the crystal structure that is in the NMR The helix is within a between residues and in the NMR In these residues are in the crystal structure with the of residues in B. The differences between the two structures can by that structural between the amino-terminal region and the core region The amino-terminal α-helix is with the of the core region and as as bonds from the side of and in the core region are the active site by with the and this the of the two that were not in the solution In addition, the interaction between the amino-terminal helix and the is by between the two of the structural data with the MerB crystal to L. C. Science. PubMed Scopus Google Scholar). The only protein with structural is the protein Biochemistry. 2006; PubMed Scopus Google Scholar), and the is only with the core region of MerB = mean square = with L. J. 2000; PubMed Scopus Google The in the by E. coli cysteines and and NMR studies demonstrated that three cysteines and are the active site (17Di Lello P. Benison G.C. Valafar H. Pitts K.E. Summers A.O. Legault P. Omichinski J.G. Biochemistry. 2004; 43: 8322-8332Crossref PubMed Scopus (30) Google Scholar). demonstrated that the highly conserved Cys-96 and Cys-159 are essential for but the with a only reduces (16Pitts K.E. Summers A.O. Biochemistry. 2002; 41: 10287-10296Crossref PubMed Scopus (65) Google Scholar). In the crystal structure, the of Cys-96 and Cys-159 are within the protein are as a of the amino-terminal α-helix in contact with the core region the between residues and are two to the of Cys-96 and Cys-159 The is by the and the is by the amino-terminal In is (17Di Lello P. Benison G.C. Valafar H. Pitts K.E. Summers A.O. Legault P. Omichinski J.G. Biochemistry. 2004; 43: 8322-8332Crossref PubMed Scopus (30) Google Scholar). of of a crystals of free MerB were soaked with organomercurials or para-hydroxymercury benzoic in the or of and These a mercury-bound as to an of MerB. In there was that either the carbon side chain or the thiol to the mercury and This is in to NMR studies where of MerB with organomercurial compounds in the of in a G.C. Lello P. Legault P. Omichinski J.G. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). The structure of mercury-bound MerB is very similar to that of the free MerB with the of the mercury and the two structures have a mean square of The mercury is to MerB in a by two from Cys-96 and Cys-159 and a The binding the mercury is from a at of and with the mercury and the of Cys-96 and with the free MerB, the active site residues are to of their cysteine to mutants and of MerB were prepared in an to generate The structures of the three mutants were to that of the MerB in the free with organomercurial we observed for organomercurial substrate or mercury product with either the C96S or C159S not In we observed mercury product in the C160S Table This that the C160S crystal and that is not required for MerB activity. These results are in with mutagenesis studies the of these mutants on MerB (16Pitts K.E. Summers A.O. Biochemistry. 2002; 41: 10287-10296Crossref PubMed Scopus (65) Google Scholar). In addition, these results that Cys-96 and Cys-159 are essential for both substrate binding and for carbon-mercury bond Asp-99 in to as a of the mechanism of MerB, Cys-96 Cys-159 and with the organomercurial to a structure the mercury that makes the carbon-mercury bond more to Analysis of the crystal structure of the mercury-bound of MerB that Asp-99 is the only acid to the mercury that can as proton with of its within and the of the side chain within of the to mercury. In addition, Asp-99 is in close proximity to the of Cys-96 in the crystal structure of the free and in an to in the step required for the prior to of the organomercurial MerB and by been demonstrated that mercury compounds to mimic MerB are to cleave organomercurial compounds (18Melnick J.G. Parkin G. Science. 2007; 317: 225-227Crossref PubMed Scopus (88) Google Scholar, M. S. E. W. A. H. J. Chem. 2004; Scopus Google Scholar). a mercury three and carbon when with and in the crystal structures of and the mercury-bound of MerB the mercury that Cys-96 and Cys-159 can with two or three from This is the that mercury-bound of MerB is in a and the is in a In this the of is in the as the of and the carbon of the carbon-mercury bond of is from of the of the with which cleaves carbon-mercury this for the role of Asp-99 in the protonolysis of carbon-mercury bonds by MerB. important from the MerB crystal structures is the of the active site within the the MerB active site to the of the product ionic mercury. mercury is highly and of mercury from MerB cleavage of the carbon-mercury bond in ionic mercury MerB to transfer ionic mercury directly to the reductase MerA for to the less toxic elemental mercury. in studies demonstrated that MerB ionic mercury to and the active site is with this mechanism G.C. Lello P. Legault P. Omichinski J.G. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). MerA with MerB for the mercury and this in the transfer release. still the substrate the active and is mercury to Based on NMR residues are of a and the 25 amino-terminal residues either or they are In the crystal the high and the of residues in these support the that these two of MerB are and Because Cys-159 is of the substrate binding the of these two a The of these to bury the active site. This of structural and in a of The of the of the but the active site MerB to cleave a of organomercurial This of structural substrate binding is not only with the and high mm for observed for MerB but also with the of organomercurial to para-hydroxymercury benzoic that can The of the mercury in the mercury-bound MerB crystal is similar to we previously observed for a in solution G.C. Lello P. Legault P. Omichinski J.G. Biochemistry. 2004; 43: PubMed Scopus Google Scholar). In this the mercury Cys-96 and two from In this structure, Cys-159 is in with the for binding to the mercury. that the is in the Cys-159 can by the Despite this there is a of the and the of the three to the mercury is the This can as step the reaction The a with two can with Cys-159 for the mercury and serves as a model for two cysteines of MerA to the mercury from MerB in a mechanism of crystal structure of the cysteine mutants of MerB to the for the three cysteines residues the active site. The MerB crystals are and in the of organomercurials a mercury-bound is In the mercury-bound Cys-96 and Cys-159 are to the mercury in a and this can either in the or of In we were to a mercury-bound with crystals of either the C96S or C159S but we were to generate a mercury-bound with crystals of the C160S These results demonstrate that Cys-96 and Cys-159 have a role in substrate binding, carbon-mercury bond cleavage, and of mercury from the active site. Asp-99 as a mechanism for chemical cleavage of carbon-mercury bonds the of the mercury by by protonolysis of the carbon-mercury bond (18Melnick J.G. Parkin G. Science. 2007; 317: 225-227Crossref PubMed Scopus (88) Google Scholar, M. S. E. W. A. H. J. Chem. 2004; Scopus Google Scholar). Although the step to the of organomercurial compounds with high the high number been the protonolysis step is to (18Melnick J.G. Parkin G. Science. 2007; 317: 225-227Crossref PubMed Scopus (88) Google Scholar). of the crystal structure of with the crystal structures of MerB that MerB cleaves the carbon-mercury bond in a similar to these chemical proposed Cys-96 the and we that the Asp-99 The of Asp-99 is in proximity to Cys-96 in the free it is to Cys-159 by Cys-159 to an organomercurial with a high Asp-99 is to the proton required for the protonolysis Asp-99 as the proton or by the of Cys-96 and this proton to the carbon moiety for In of the MerB isolated from bacteria resistant to Asp-99 is conserved for sequence where it is by a this a of substrate M. T. G. 1999; PubMed Scopus Google Scholar). NMR studies with a MerB demonstrated that this protein is and not cleave carbon-mercury bonds the same as MerB not This to the of the MerB active site and the of Asp-99 in the catalytic mechanism of MerB. on the crystal structures of MerB, we a model for the mechanism of carbon-mercury bond cleavage This mechanism is to previously proposed (15Begley T.P. Walts A.E. Walsh C.T. Biochemistry. 1986; 25: 7192-7200Crossref PubMed Scopus (89) Google Scholar, K.E. Summers A.O. Biochemistry. 2002; 41: 10287-10296Crossref PubMed Scopus (65) Google Scholar, J.G. Parkin G. Science. 2007; 317: 225-227Crossref PubMed Scopus (88) Google Scholar, M. S. E. W. A. H. J. Chem. 2004; Scopus Google Scholar, E. J. Chem. Scopus Google Scholar). In it is in with the that a present in MerB as the proton for the carbon-mercury bond cleavage E. J. Chem. Scopus Google Scholar). the crystal structures of the in which the cysteine residues and Asp-99 as the residue that serves as the proton In an thiol is to the organomercurial in the of the The step MerB is the of Cys-96 by Cys-96 attacks the organomercurial and Cys-159 attacks the mercury and the These two in the of a species with mercury This structural to the binding between the and the amino-terminal helix The mercury is in proximity to which serves to the carbon-mercury bond and the that is by This results in bond cleavage and the retention of the ionic mercury in the active site. The ionic mercury is directly to MerA for In this MerB and MerA the toxic organomercurial and ionic mercury species at times and their with In this study, we the crystal structure of MerB in its free and mercury-bound These structures provide important that is essential to ongoing attempts to exploit the of the mer system in the bioremediation of methylmercury. Parkin for on the was out in at the National Synchrotron Light National Laboratory, which is by the of of and of with

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