The Roles of Thiols in the Bacterial Organomercurial Lyase (MerB)

Abstract

The bacterial plasmid-encoded organomercurial lyase, MerB (EC 4.99.1.2), catalyzes the protonolysis of organomercury compounds yielding Hg(II) and the corresponding protonated hydrocarbon. A small, soluble protein with no known homologues, MerB is widely distributed among eubacteria in three phylogenetically distinct subfamilies whose most prominent motif includes three conserved cysteine residues. We found that the 212-residue MerB encoded by plasmid R831b is a cytosolic enzyme, consistent with its high thiol requirement in vitro. MerB is inhibited by the nonphysiological dithiol DTT but uses the physiological thiols, glutathione and cysteine, equally well. Highly conserved Cys96 and Cys159 are essential for activity, whereas weakly conserved Cys160 is not. Proteins mutant in highly conserved Cys117 are insoluble. All MerB cysteines are DTNB-reactive in native and denatured states except Cys117, which fails to react with DTNB in the native form, suggesting it is buried. Mass spectrometric analysis of trypsin fragments of reduced proteins treated with N-ethylmaleimide or iodoacetamide revealed that all cysteines form covalent adducts and remain covalently modifiable even when exposed to 1:1 PHMB prior to treatment with NEM or IAM. Stable PHMB adducts were also observed on all cysteines in mutant proteins, suggesting rapid exchange of PHMB among the remaining protein thiols. However, PHMB exposure of reduced wild-type MerB yielded only Hg adducts on the Cys159/Cys160 peptide, suggesting a trapped reaction intermediate. Using HPLC to follow release of benzoic acid from PHMB, we confirmed that fully reduced wild-type MerB and mutant C160S can carry out a single protonolysis without exogenous thiols. On the basis of the foregoing we refine the previously proposed S(E)2 mechanism for protonolysis by MerB.

Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation

“…In the organomercurial lyase protein MerB, two vicinal cysteines act in coordination with a third cysteine to have maximal function (56). Mutation of one of the vicinal cysteines in this system led to only a slight reduction in activity (57). Future studies may determine whether Cys73 of HgcB functions in conjunction with either of the vicinal cysteines for electron movement, Hg binding, or structural integrity.…”

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

“…The MerB mutants (C96S MerB, C159S MerB, and C160S MerB) were prepared by site-directed mutagenesis of plasmid pQZB1 (16). Wild-type MerB was expressed and purified as described previously (16,17).…”

“…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 (16). 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.…”

Effect of Inorganic and Organic Ligands on the Bioavailability of Methylmercury as Determined by Using a mer-lux Bioreporter

“…The full-length sequence of organomercurial lyase (merB) was PCR amplified from the E. coli plasmid pQZB1 (23). PCR was performed using the reported method (36) with the primer set merbEXF1N-merbEXR1 (Table 1), where the lowercase letters are the inserted sequences containing restriction enzyme sites (BamHI and XbaI) and the GAGA sequence protects the restriction enzyme sites.…”