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Mechanistic pathways of mercury removal from the organomercurial lyase active site.

Silva PJ, Rodrigues V - PeerJ (2015)

Bottom Line: Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+).Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction.Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

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Affiliation: FP-ENAS/Fac. de Ciências da Saúde, Universidade Fernando Pessoa , Porto , Portugal.

ABSTRACT
Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

No MeSH data available.


Numerical simulations of different portions of the MerB reaction mechanism in the presence of glutathione, using reaction rates derived from the energies computed by our quantum chemical computations.(A) Thiolate-based pathways only; (B) thiolate-based pathways + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero; (C) combined operation of thiolate-based pathways and Asp-assisted thiol addition pathways; (D) thiolate-based pathways + Cys-assisted thiol addition + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero. Glutathione concentrations are: 0.5 mM (blue), 1.5 mM (yellow), 2.5 mM (red), 5 mM (green), and 10 mM (purple)
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fig-9: Numerical simulations of different portions of the MerB reaction mechanism in the presence of glutathione, using reaction rates derived from the energies computed by our quantum chemical computations.(A) Thiolate-based pathways only; (B) thiolate-based pathways + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero; (C) combined operation of thiolate-based pathways and Asp-assisted thiol addition pathways; (D) thiolate-based pathways + Cys-assisted thiol addition + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero. Glutathione concentrations are: 0.5 mM (blue), 1.5 mM (yellow), 2.5 mM (red), 5 mM (green), and 10 mM (purple)

Mentions: Extensive experimental analysis of the reaction of Hg-bound MerB with glutathione or the physiological partner (Hong et al., 2010) has shown that MerA is able to effect complete metal removal even at very low concentrations (50 µM), whereas concentrations of monothiols below 10 mM afford only partial protein demetallation. Numerical simulation of the complete reaction mechanism described in this work (Fig. 8) reveals a very good agreement with experiment, provided that a protonated thiol is prevented from performing the initial attack on the mercury ion (Fig. 9A and 9B): operation of the Asp-assisted pathway (either alone or in concert with other pathways) would always lead to complete removal of mercury from the MerB active site (Fig. 9C) due to the high exergonicity of the initial formation of the Asp-protonated form of Int1 intermediate (Fig. 3A and Table 2). Simultaneous operation of the Cys-assisted pathways would in turn allow the C96-bound Int3′ intermediate (formed mainly in the thiolate pathway, which has a more exergonic first reaction than the Cys-assisted thiol attack pathway) to be diverted through thiolate loss (reaction k6 in Fig. 8) to the Cys-assisted pathway, yielding a complex kinetic profile which ultimately leads to total mercury removal from MerB (Fig. 9D). In turn, setting the reaction rate of the k5 and k6 steps to zero (i.e., preventing the conversion of Int2 (C96-bound) into Int3′ (C96-bound, and vice-versa)), while keeping the thiolate-only pathway and the rest of the Cys-assisted pathway operative yields a kinetic profile indistinguishable from that of the thiolate-only pathway. Interestingly, identical kinetic simulations using the DTT (which is a known inhibitor of MerB) failed to show any inhibition. The agreement of our model with the experimental observations therefore requires that the formation of Int1 (protonated Asp) (Fig. 8, reaction k15/k16), the conversion of Int2 (C96-bound) into Int3′ (C96-bound) (Fig. 8, reaction k5/k6), and the release of the Hg-DTT complex from the active site, which are predicted by our small-model QM computations to be thermodynamically and kinetically feasible, are prevented in the enzyme, most likely due to the intervention of steric factors arising from the rest of the protein. The proposed role of steric factors in the overall kinetic profile of MerB is consistent with other experimental observation: for example, though the trigonal complex of Hg bound by both sulfur atoms of DTT and by Cys96 is long-lived in the absence of added thiols, Hg can be removed after a few minutes of incubation with MerA or with incubation with very high concentrations of glutathione (Benison et al., 2004). Since the chemically reactive portion in all these MerB co-substrates is the same, this implies that the differences in behavior should not be based in purely electronic factors but in the presence of intermolecular interactions between Hg-DTT and MerB (but not between Hg-MerA and MerB, or between Hg-glutathione and MerB) which prevent its exit from the active site. Indeed, preliminary experimental evidence (J Omichinski, pers. comm., 2015) suggests that the N-terminal portion of MerB is responsible for trapping the Hg-DTT complex and the observed partial inhibition of MerB activity by DTT.


Mechanistic pathways of mercury removal from the organomercurial lyase active site.

Silva PJ, Rodrigues V - PeerJ (2015)

Numerical simulations of different portions of the MerB reaction mechanism in the presence of glutathione, using reaction rates derived from the energies computed by our quantum chemical computations.(A) Thiolate-based pathways only; (B) thiolate-based pathways + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero; (C) combined operation of thiolate-based pathways and Asp-assisted thiol addition pathways; (D) thiolate-based pathways + Cys-assisted thiol addition + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero. Glutathione concentrations are: 0.5 mM (blue), 1.5 mM (yellow), 2.5 mM (red), 5 mM (green), and 10 mM (purple)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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fig-9: Numerical simulations of different portions of the MerB reaction mechanism in the presence of glutathione, using reaction rates derived from the energies computed by our quantum chemical computations.(A) Thiolate-based pathways only; (B) thiolate-based pathways + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero; (C) combined operation of thiolate-based pathways and Asp-assisted thiol addition pathways; (D) thiolate-based pathways + Cys-assisted thiol addition + conversions to intermediates of the Asp-assisted pathway. Rates of reactions k15/k16 set to zero. Glutathione concentrations are: 0.5 mM (blue), 1.5 mM (yellow), 2.5 mM (red), 5 mM (green), and 10 mM (purple)
Mentions: Extensive experimental analysis of the reaction of Hg-bound MerB with glutathione or the physiological partner (Hong et al., 2010) has shown that MerA is able to effect complete metal removal even at very low concentrations (50 µM), whereas concentrations of monothiols below 10 mM afford only partial protein demetallation. Numerical simulation of the complete reaction mechanism described in this work (Fig. 8) reveals a very good agreement with experiment, provided that a protonated thiol is prevented from performing the initial attack on the mercury ion (Fig. 9A and 9B): operation of the Asp-assisted pathway (either alone or in concert with other pathways) would always lead to complete removal of mercury from the MerB active site (Fig. 9C) due to the high exergonicity of the initial formation of the Asp-protonated form of Int1 intermediate (Fig. 3A and Table 2). Simultaneous operation of the Cys-assisted pathways would in turn allow the C96-bound Int3′ intermediate (formed mainly in the thiolate pathway, which has a more exergonic first reaction than the Cys-assisted thiol attack pathway) to be diverted through thiolate loss (reaction k6 in Fig. 8) to the Cys-assisted pathway, yielding a complex kinetic profile which ultimately leads to total mercury removal from MerB (Fig. 9D). In turn, setting the reaction rate of the k5 and k6 steps to zero (i.e., preventing the conversion of Int2 (C96-bound) into Int3′ (C96-bound, and vice-versa)), while keeping the thiolate-only pathway and the rest of the Cys-assisted pathway operative yields a kinetic profile indistinguishable from that of the thiolate-only pathway. Interestingly, identical kinetic simulations using the DTT (which is a known inhibitor of MerB) failed to show any inhibition. The agreement of our model with the experimental observations therefore requires that the formation of Int1 (protonated Asp) (Fig. 8, reaction k15/k16), the conversion of Int2 (C96-bound) into Int3′ (C96-bound) (Fig. 8, reaction k5/k6), and the release of the Hg-DTT complex from the active site, which are predicted by our small-model QM computations to be thermodynamically and kinetically feasible, are prevented in the enzyme, most likely due to the intervention of steric factors arising from the rest of the protein. The proposed role of steric factors in the overall kinetic profile of MerB is consistent with other experimental observation: for example, though the trigonal complex of Hg bound by both sulfur atoms of DTT and by Cys96 is long-lived in the absence of added thiols, Hg can be removed after a few minutes of incubation with MerA or with incubation with very high concentrations of glutathione (Benison et al., 2004). Since the chemically reactive portion in all these MerB co-substrates is the same, this implies that the differences in behavior should not be based in purely electronic factors but in the presence of intermolecular interactions between Hg-DTT and MerB (but not between Hg-MerA and MerB, or between Hg-glutathione and MerB) which prevent its exit from the active site. Indeed, preliminary experimental evidence (J Omichinski, pers. comm., 2015) suggests that the N-terminal portion of MerB is responsible for trapping the Hg-DTT complex and the observed partial inhibition of MerB activity by DTT.

Bottom Line: Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+).Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction.Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

View Article: PubMed Central - HTML - PubMed

Affiliation: FP-ENAS/Fac. de Ciências da Saúde, Universidade Fernando Pessoa , Porto , Portugal.

ABSTRACT
Bacterial populations present in Hg-rich environments have evolved biological mechanisms to detoxify methylmercury and other organometallic mercury compounds. The most common resistance mechanism relies on the H(+)-assisted cleavage of the Hg-C bond of methylmercury by the organomercurial lyase MerB. Although the initial reaction steps which lead to the loss of methane from methylmercury have already been studied experimentally and computationally, the reaction steps leading to the removal of Hg(2+) from MerB and regeneration of the active site for a new round of catalysis have not yet been elucidated. In this paper, we have studied the final steps of the reaction catalyzed by MerB through quantum chemical computations at the combined MP2/CBS//B3PW91/6-31G(d) level of theory. While conceptually simple, these reaction steps occur in a complex potential energy surface where several distinct pathways are accessible and may operate concurrently. The only pathway which clearly emerges as forbidden in our analysis is the one arising from the sequential addition of two thiolates to the metal atom, due to the accumulation of negative charges in the active site. The addition of two thiols, in contrast, leads to two feasible mechanistic possibilities. The most straightforward pathway proceeds through proton transfer from the attacking thiol to Cys159 , leading to its removal from the mercury coordination sphere, followed by a slower attack of a second thiol, which removes Cys96. The other pathway involves Asp99 in an accessory role similar to the one observed earlier for the initial stages of the reaction and affords a lower activation enthalpy, around 14 kcal mol(-1), determined solely by the cysteine removal step rather than by the thiol ligation step. Addition of one thiolate to the intermediates arising from either thiol attack occurs without a barrier and produces an intermediate bound to one active site cysteine and from which Hg(SCH3)2 may be removed only after protonation by solvent-provided H3O(+). Thiolate addition to the active site (prior to any attack by thiols) leads to pathways where the removal of the first cysteine becomes the rate-determining step, irrespective of whether Cys159 or Cys96 leaves first. Comparisons with the recently computed mechanism of the related enzyme MerA further underline the important role of Asp99 in the energetics of the MerB reaction. Kinetic simulation of the mechanism derived from our computations strongly suggests that in vivo the thiolate-only pathway is operative, and the Asp-assisted pathway (as well as the conversion of intermediates of the thiolate pathway into intermediates of the Cys-assisted pathway) is prevented by steric factors absent from our model and related to the precise geometry of the organomercurial binding-pocket.

No MeSH data available.