Journal
BIOCHEMISTRY
Volume 55, Issue 7, Pages 1070-1081Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.biochem.5b01298
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Funding
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- National Science Foundation [CHE-1308598]
- Ministry of Higher Education and scientific research in Egypt (cultural affairs and missions sector)
- U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences [DE-AC02-98CH10886]
- Canadian Light Source (CLS) - NSERC
- National Research Council Canada
- Canadian Institutes of Health Research
- Province of Saskatchewan
- Western Economic Diversification Canada
- University of Saskatchewan
- Division Of Chemistry
- Direct For Mathematical & Physical Scien [1308598] Funding Source: National Science Foundation
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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.
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