NMR Structural Studies Reveal a Novel Protein Fold for MerB, the Organomercurial Lyase Involved in the Bacterial Mercury Resistance System<sup>,</sup>

Lello, Paola Di; Benison, Gregory; Valafar, Homayoun; Pitts, Keith; Summers, Anne O.; Legault, Pascale; Omichinski, James G.

doi:10.1021/bi049669z

Cited by 38 publications

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“…Expression of Recombinant Proteins-The sequence encoding MerB from E. coli plasmid R831b was cloned as described previously (17). The MerB mutants (C96S MerB, C159S MerB, and C160S MerB) were prepared by site-directed mutagenesis of plasmid pQZB1 (16).…”

Section: Methodsmentioning

confidence: 99%

“…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). Prior to crystallization, proteins were dialyzed (see supplemental methods).…”

Section: Methodsmentioning

confidence: 99%

“…The MerB crystal structure is a homodimer in the asymmetric unit, although MerB in solution has been shown to be monomeric ( Fig. 1) (14,17). A 2-fold non-crystallographic symmetry exists between the two subunits (A and B) present in the crystal.…”

Section: Crystallization Conditions and Structure Determination-mentioning

confidence: 99%

“…The Active Site Is Buried in the Crystal Structure-MerB encoded by E. coli (R831b) contains four cysteines (Cys-96, Cys-117, Cys-159, and Cys-160), and NMR studies demonstrated that three cysteines (Cys-96, Cys-159, and Cys-160) are positioned near the active site (17). Point mutations demonstrated that the highly conserved Cys-96 and Cys-159 are essential for enzymatic activity, but replacing the non-conserved Cys-160 with a serine only moderately reduces activity (16).…”

Section: Crystallization Conditions and Structure Determination-mentioning

confidence: 99%

“…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 (17). Recently model compounds have been shown to cleave carbon-mercury bonds using only * This work was supported in part by grants from the Natural Science and Engineering Council of Canada (to J. G. O. and J. S.) and Canadian Institutes of Health Research (CIHR) (to J.…”

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

Lafrance‐Vanasse¹,

Lefebvre

Lello³

et al. 2009

Journal of Biological Chemistry

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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 ␣-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.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Crystallization Conditions and Structure Determination-mentioning

confidence: 99%

Section: Crystallization Conditions and Structure Determination-mentioning

confidence: 99%

mentioning

confidence: 96%

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

Lafrance‐Vanasse¹,

Lefebvre

Lello³

et al. 2009

Journal of Biological Chemistry

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The Organomercurial LyaseMerB

Lello

Lafrance‐Vanasse

Omichinski

2004

Handbook of Metalloproteins

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Mercury is introduced into the environment either from natural occurrences or from human activities, and consumption of mercury‐contaminated fish poses a serious human health issue. The three inorganic forms of mercury are elemental mercury, mercurous compounds, and mercuric compounds, while the most abundant organic form is methylmercury. Owing to its ability to permeate membranes and accumulate in organisms, methylmercury is more toxic than ionic mercury. Mercury‐resistant bacteria have developed a two enzyme system to convert both Hg (II) and methylmercury to the less toxic elemental mercury. The first enzyme is an organomercurial lyase (MerB) and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon–mercury bond on a wide range of organomercurials, including methylmercury, resulting in a reduced carbon compound and ionic mercury. The cleavage of the carbon–mercury bond and the formation of the electrophile‐carbon bond are concerted (S E 2). Structural studies demonstrated that MerB contains a unique fold and that significant conformational changes occur on binding of organomercurial substrates. On the basis of mutagenesis, structural, and computational studies, two cysteines and an aspartic acid residue in the active site are known to play key roles in the cleavage of the carbon–mercury bond.

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The Organomercurial LyaseMerB

Lello

Lafrance‐Vanasse

Omichinski

2011

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Enzyme; lyase; EC 4.99.1.2; an alkylmercury lyase or organomercurial lyase commonly known as MerB.MerB catalyzes the protonolysis of the carbon-mercury bond in organomercurials, yielding ionic mercury and the corresponding hydrocarbon (see Scheme 1). MerB is active on a large variety of substrates ranging from simple alkyl compounds like methylmercury, which is the most naturally abundant organomercurial, to polyaromatic heterocyclic compounds like merbromin. 3,4 MerB also catalyzes the cleavage of the carbon-tin bond in select organostannanes like tetravinyltin, but in a less efficient manner. 5 O C C U R R E N C EThe organomercurial lyase MerB is a cytosolic protein found in gram-positive and gram-negative bacterial strains that display resistance to both ionic mercury and organomecurial compounds. [6][7][8][9] The mercury resistance is due to a dedicated set of genes that are regulated together by the mer operon. 10,11 The mer operon occurs in most cases on transferable genetic elements such as transposons or plasmids, and it is not part of the native genome. The activation of the mer operon occurs only in response to the presence of compounds containing mercury, which bind to the DNAbound MerR and activate expression of the mer operon. 12 The composition of the mer operon can vary dramatically between different bacterial strains, but resistance to organomercurial requires the presence of two enzymes, the lyase MerB and the mercuric reductase MerA. 9 B I O L O G I C A L F U N C T I O NMerB belongs to a group of proteins that confers organomercurials with resistance to specific bacteria containing the mer operon. 9,13 The mercury detoxification 3D Structure Ribbon representation of the mercury-bound form of the organomercurial lyase MerB. PDB code: 3F0O. The core domain of the protein is colored in dark blue, the amino-terminal region of the protein is colored in teal, and the mercury ion is represented as a gray sphere. This representation and all other structural representations were prepared using PyMOL 1 unless noted otherwise.

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NMR Structural Studies Reveal a Novel Protein Fold for MerB, the Organomercurial Lyase Involved in the Bacterial Mercury Resistance System^,

Cited by 38 publications

References 56 publications

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

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

The Organomercurial LyaseMerB

The Organomercurial LyaseMerB

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NMR Structural Studies Reveal a Novel Protein Fold for MerB, the Organomercurial Lyase Involved in the Bacterial Mercury Resistance System,

Cited by 38 publications

References 56 publications

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

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

The Organomercurial LyaseMerB

The Organomercurial LyaseMerB

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NMR Structural Studies Reveal a Novel Protein Fold for MerB, the Organomercurial Lyase Involved in the Bacterial Mercury Resistance System^,