Structural and Biochemical Characterization of a Copper-Binding Mutant of the Organomercurial Lyase MerB: Insight into the Key Role of the Active Site Aspartic Acid in Hg–Carbon Bond Cleavage and Metal Binding Specificity

Abstract

In bacterial resistance to mercury, the organomercurial lyase (MerB) plays a key role in the detoxification pathway through its ability to cleave Hg-carbon bonds. Two cysteines (C96 and C159; Escherichia coli MerB numbering) and an aspartic acid (D99) have been identified as the key catalytic residues, and these three residues are conserved in all but four known MerB variants, where the aspartic acid is replaced with a serine. To understand the role of the active site serine, we characterized the structure and metal binding properties of an E. coli MerB mutant with a serine substituted for D99 (MerB D99S) as well as one of the native MerB variants containing a serine residue in the active site (Bacillus megaterium MerB2). Surprisingly, the MerB D99S protein copurified with a bound metal that was determined to be Cu(II) from UV-vis absorption, inductively coupled plasma mass spectrometry, nuclear magnetic resonance, and electron paramagnetic resonance studies. X-ray structural studies revealed that the Cu(II) is bound to the active site cysteine residues of MerB D99S, but that it is displaced following the addition of either an organomercurial substrate or an ionic mercury product. In contrast, the B. megaterium MerB2 protein does not copurify with copper, but the structure of the B. megaterium MerB2-Hg complex is highly similar to the structure of the MerB D99S-Hg complexes. These results demonstrate that the active site aspartic acid is crucial for both the enzymatic activity and metal binding specificity of MerB proteins and suggest a possible functional relationship between MerB and its only known structural homologue, the copper-binding protein NosL.

Structural and Biochemical Characterization of Organotin and Organolead Compounds Binding to the Organomercurial Lyase MerB Provide New Insights into Its Mechanism of Carbon–Metal Bond Cleavage

“…47–49 Finally, substituting serine in place of D99 in E. coli MerB results in a mutant protein that sequesters Cu II when expressed in bacteria and has significantly lower catalytic activity. 50 Taken together with our current results on organotin and organolead compounds, initial binding to D99 is important not only for the catalytic activity of MerB, but also for metal selectivity.…”

Expanded Diversity and Phylogeny of mer Genes Broadens Mercury Resistance Paradigms and Reveals an Origin for MerA Among Thermophilic Archaea

“…The 13 MerB-like 99Ser variants were identified in genomes that all contain at least one MerB with the aforementioned canonical sequence signatures; these genomes belonged to members of the bacterial phylum Firmicutes. Previous structural and biochemical characterization of representatives of the 99Ser MerB-like variants ( Wahba et al, 2016 ) has shown that they have expanded range of affinities for metals relative to canonical MerB. Specifically, this variant has an affinity to bind Cu(II) that is displaced by Hg(II) when present.…”

“…Hg(II) is a product of MerB-dependent demethylation of MeHg, and MerB binds Hg(II) so that it cannot diffuse into the cytoplasm; MerA then extracts the metal for reduction ( Wahba et al, 2016 ). The physiological role of these variant enzyme with an affinity for other divalent metals is thus not well understood especially when a native MerB 99Ser, in B. megaterium MerB2, does not bind Cu(II) ( Wahba et al, 2016 ). However, it is possible that they function as more generalized organic metalloid detoxification enzymes than canonical MerB.…”

Shining light on recent advances in microbial mercury cycling

“…FACETS | 2018 | 3: 858-879 | DOI: 10.1139/facets-2018-0015 861 facetsjournal.com Recent work on MerB has shown that conserved cysteine and aspartic acid residues are essential for releasing Hg from MerB's active site following the cleaving of the methyl group (Silva and Rodrigues 2015). When a serine residue was present in place of aspartic acid, MerB lost its specificity for MeHg and was also able to bind copper (Cu), although resupplying MerB with Hg successfully removed Cu from the enzyme's active site (Wahba et al 2016). These findings demonstrate that MeHg can outcompete Cu for the binding site on MerB, but also highlight functional parallels that may exist between RD and Cu homeostasis that merit further investigation.…”