Structure and mechanism of merB
Benison, Gregory Charles
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The three major naturally occurring forms of mercury- elemental, ionic, and organicare widely distributed throughout the biosphere. All three forms are constantly interconverted by a variety of abiotic and biological mechanisms. Microbial mechanisms dominate the conversion of ionic mercury to organic mercury compounds, especially methyl mercury. Some microorganisms are also capable of transforming ionic or organic mercury into elemental mercury. This capability constitutes a detoxification mechanism due to the lower toxicity of elemental mercury relative to ionic or organic mercury. Organic and ionic mercury resistance is due to a dedicated set of plasmid-encoded mercury resistance genes (the mer operon.) merR is a regulatory gene. merP and merT are involved in mercury binding and transport. merA encodes for a reductase which reduces ionic mercury to elemental mercury using NADPH. merB encodes for an organomercurial lyase, which converts organomercurials into ionic mercury and a reduced organic product. In this study, the preparation of several mercury-containing MerB complexes is described. Of the complexes prepared, the most stable was identified by Nuclear Magnetic Resonance (NMR) and Extended X-Ray Absorption Fine Structure (EXAFS) spectroscopy as a complex of MerB, a mercuric ion, and one molecule of dithiothreitol (DTT). The mercuric ion is in a trigonal geometry, coordinated by both sulfur atoms of DTT as well as the sulfur atom of C96 from MerB. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, was demonstrated by NMR and dialysis. The structure of the MerB/Hg/DTT complex was determined by NMR to 1.11 °A resolution. Due to the toxicity of mercuric ions, some mercury resistance proteins may employ a mechanism in which mercuric ions pass directly between the proteins, without diffusing through the cytosol. In order to investigate the possibility of direct transfer between MerB and MerA, the MerB/Hg/DTT complex was used as a substrate for MerA in an enzyme buffering test. The observed MerA activity was higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA, which suggests that the mercuric ion can be directly transferred between the two enzymes.