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

Cited by 57 publications

(87 citation statements)

References 37 publications

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“…The possibility of vicinal cysteines (Cys94 and Cys95) working together with a separate cysteine (Cys73) to act on a Hg substrate has precedence. 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).…”

Section: Discussionmentioning

confidence: 99%

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

Smith

Bridou

Johs

et al. 2015

Appl Environ Microbiol

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f Methylmercury is a potent neurotoxin that is produced by anaerobic microorganisms from inorganic mercury by a recently discovered pathway. A two-gene cluster, consisting of hgcA and hgcB, encodes two of the proteins essential for this activity. hgcA encodes a corrinoid protein with a strictly conserved cysteine proposed to be the ligand for cobalt in the corrinoid cofactor, whereas hgcB encodes a ferredoxin-like protein thought to be an electron donor to HgcA. Deletion of either gene eliminates mercury methylation by the methylator Desulfovibrio desulfuricans ND132. Here, site-directed mutants of HgcA and HgcB were constructed to determine amino acid residues essential for mercury methylation. Mutations of the strictly conserved residue Cys93 in HgcA, the proposed ligand for the corrinoid cobalt, to Ala or Thr completely abolished the methylation capacity, but a His substitution produced measurable methylmercury. Mutations of conserved amino acids near Cys93 had various impacts on the methylation capacity but showed that the structure of the putative "cap helix" region harboring Cys93 is crucial for methylation function. In the ferredoxin-like protein HgcB, only one of two conserved cysteines found at the C terminus was necessary for methylation, but either cysteine sufficed. An additional, strictly conserved cysteine, Cys73, was also determined to be essential for methylation. This study supports the previously predicted importance of Cys93 in HgcA for methylation of mercury and reveals additional residues in HgcA and HgcB that facilitate the production of this neurotoxin. M ethylmercury (MeHg), a neurotoxin present in the environment, is a significant risk to human health in many regions of the world (1). Sources of the mercury (Hg) substrate from which MeHg is derived are numerous and include both natural and anthropogenic sources (2, 3). Currently, only anaerobic microbes are known to produce MeHg (4), predominantly in sediments of aquatic environments. Once MeHg is produced, it accumulates in the aquatic food chain, becoming concentrated in top predators (5). Human exposure to MeHg results from eating those predators, for example, in marine food webs, shark, swordfish, albacore tuna, and eel. Once in the body, MeHg passes through the intestinal epithelium, usually complexed with thiol groups in proteins (6). Eventually, MeHg buildup in humans can lead to neuropathies in adults and developmental disorders in children exposed in utero (2).Anaerobic Deltaproteobacteria are thought to be the major contributors of MeHg found in the environment (7, 8), although potentially significant contributions by methanogens and fermenting bacteria have recently been discovered (9-11). In 2013, we showed that the proteins encoded by two genes in two bacteria of the Deltaproteobacteria phylum, Desulfovibrio desulfuricans ND132 and Geobacter sulfurreducens PCA, are essential for the conversion of Hg(II) to MeHg (12). That study was stimulated by the work of Choi and colleagues (13-15) implicating a 40-kDa corrinoid protein i...

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Section: Discussionmentioning

confidence: 99%

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

Smith

Bridou

Johs

et al. 2015

Appl Environ Microbiol

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“…Active-site cluster models of MerB were generated using an X-ray crystal structure of the Hg(II)-product complex [24]. The side chains of Cys96, Cys159, Asp99 were included, as were Hg(II) and a single ordered water molecule observed in the crystal structure.…”

Section: Methods For Dft Calculationsmentioning

confidence: 99%

“…This intermediate step enables both cysteines to coordinate with methylmercury. Upon coordination, Asp99 then protonates the CH 3 À leaving group and yields the Hg-C cleavage products, Hg(II) and methane [4,24]. Several computational studies using DFT and ab initio methods have been conducted to address mercury-carbon bond cleavage reactions [26][27][28][29][30], but these have failed to answer key questions related to the specific mechanism of MerB because no X-ray crystal structures were yet available.…”

Section: Introductionmentioning

confidence: 99%

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Mercury Detoxification by Bacteria: Simulations of Transcription Activation and MercuryCarbon Bond Cleavage

Guo

Parks

Johs

et al. 2011

Modeling of Molecular Properties

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“…The enzyme itself is relatively small at around 24kDa, having been structurally characterized through crystallography, including various mutants, which have helped deduce the functional domain, key amino residues, and mechanistic architecture [11,12]. The may give this enzyme the ability to remain functional while immobilized on an extracellular solid substrate.…”

Section: +mentioning

confidence: 99%

Immobilized Organo-Mercurial Lyase on Zeolite Using a Solid Binding Peptide

McCarthy¹,

Edwards²

2018

IJEBB

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Methylmercury (MeHg) compounds can form naturally, are highly toxic, and of concern because of their tendency to bio-accumulate. Certain bacteria have evolved mechanisms that can tolerate MeHg by first demethylating MeHg compounds, before further processing. Drawing inspiration from this demethylation mechanism controlled by a single organo-mercurial lyase in a protonolysis reaction, this research uses a recombinant gene that produces this lyase plus an additional polypeptide that selectively binds to zeolite particles, effectively tethering the enzyme to the solid substrate. This work is part of a broader attempt to create a fixed bed reactor for de-methylation of MeHg. Enzyme immobilization was achieved using a solid binding peptide (SBP) with high affinity for faujasite zeolite (FZ), the choice of binding substrate in the present work. The lyase is coded for by the merB gene, and a sequence with highly conserved active site homology was obtained from E.coli plasmid R8361b. The SBP plus merB sequence was designed such that the SBP was positioned either on the N or C-terminal of the construct. The DNA was synthesized commercially, and expressed in E.coli (BL21DE3 Star) using pET100® vector. Sanger sequencing was used to confirm construct in transformed cells using standard T7 oligos. Expression was lactose induced, and SDS-PAGE electrophoresis was used to confirm protein production and size. LC-MS/MS and sequence bio-analytics confirmed peptide sequence. Silica binding assays using SDS-PAGE confirmed binding of the enzyme to the silica substrate. Enzyme functionality results using a non-methylated mercuric compound were inconclusive, however the enzyme has not been assessed using MeHg compounds at this stage.

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

Cited by 57 publications

References 37 publications

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

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

Mercury Detoxification by Bacteria: Simulations of Transcription Activation and MercuryCarbon Bond Cleavage

Immobilized Organo-Mercurial Lyase on Zeolite Using a Solid Binding Peptide

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