Mechanism of Hg−C Protonolysis in the Organomercurial Lyase MerB

Abstract

Demethylation is a key reaction in global mercury cycling. The bacterial organomercurial lyase, MerB, catalyzes the demethylation of a wide range of organomercurials via Hg-C protonolysis. Two strictly conserved cysteine residues in the active site are required for catalysis, but the source of the catalytic proton and the detailed reaction mechanism have not been determined. Here, the two major proposed reaction mechanisms of MerB are investigated and compared using hybrid density functional theory calculations. A model of the active site was constructed from an X-ray crystal structure of the Hg(II)-bound MerB product complex. Stationary point structures and energies characterized for the Hg-C protonolysis of methylmercury rule out the direct protonation mechanism in which a cysteine residue delivers the catalytic proton directly to the organic leaving group. Instead, the calculations support a two-step mechanism in which Cys96 or Cys159 first donates a proton to Asp99, enabling coordination of two thiolates with R-Hg(II). At the rate-limiting transition state, Asp99 protonates the nascent carbanion in a trigonal planar, bis thiol-ligated R-Hg(II) species to cleave the Hg-C bond and release the hydrocarbon product. Reactions with two other substrates, vinylmercury and cis-2-butenyl-2-mercury, were also modeled, and the computed activation barriers for all three organomercurial substrates reproduce the trend in the experimentally observed enzymatic reaction rates. Analysis of atomic charges in the rate-limiting transition state structure using Natural Population Analysis shows that MerB lowers the activation free energy in the Hg-C protonolysis reaction by redistributing electron density into the leaving group and away from the catalytic proton.

A C⋅As lyase for degradation of environmental organoarsenical herbicides and animal husbandry growth promoters

“…MerB is a C·Hg lyase that confers resistance to organomercurials (33). Although superficially similar to ArsI, MerB is an unrelated enzyme with a different reaction mechanism (34). Similarly, merB is usually associated with merA, and mer operons that include merB are mostly broad-spectrum resistances to inorganic and organomercurials (35,36).…”

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

“…The mechanistic function of the merB product has not been fully elucidated, however it is highly likely to function as proposed by Parks et.al., [9]. 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 overall microbial strategy ofen involves the intracellular reduction of Hg 2+ -> Hg 0 by a mercuric reductase enzymatic reaction, whereby Hg 0 then naturally volatilizes and passively diffuses from the cell and out of the immediate surrounds into the atmosphere [8]. Where it is necessary for microbes to deal with MeHg, the reduction reaction is preceded by MeHg intracellular interaction with an organo-mercurial lyase, which cleaves the C-Hg bond through a protonolysis reaction [9], producing CH 4 …”

Understanding Enzyme Catalysis Using Computer Simulation

“…160 Mercury is toxic to living organisms and has no known biological function. Organomercurial species such as methylmercury, [CH 3 Hg(II)] + , bioaccumulate in living organisms due to their high affinity for thiols and other functional groups in vivo.…”

“…The quantum chemical cluster approach was used to study the Hg-C cleavage, or protonolysis, reaction catalyzed by MerB in ref 160. In particular, the aim was to determine which, if any, of the mechanisms proposed previously in the literature was correct, and also to determine how MerB achieves its rate enhancement.…”