The mercuric and organomercurial detoxifying enzymes from a plasmid-bearing strain of Escherichia coli.
Two separate enzymes, which determine resistance to inorganic mercury and organomercurials, have been purified from the plasmid-bearing Escherichia coli strain J53-1(R831). The mercuric reductase that reduces Hg2+ to volatile Hg0 was purified about 240-fold from the 160,000 X g supernatant of French press disrupted cells. This enzyme contains bound FAD, requires NADPH as an electron donor, and requires the presence of a sulfhydryl compound for activity. The reductase has a Km of 13 micron HgCl2, a pH optimum of 7.5 in 50 mM sodium phosphate buffer, an isoelectric point of 5.3, a Stokes radius of 50 A, and a molecular weight of about 180,000. The subunit molecular weight, determined by gel electrophoresis in the presence of sodium dodecyl sulfate, is about 63,000 +/- 2,000. These results suggest that the native enzyme is composed of three identical subunits. The organomercurial hydrolase, which breaks the mercury-carbon bond in compounds such as methylmercuric chloride, phenylmercuric acetate, and ethylmercuric chloride, was purified about 38-fold over the starting material. This enzyme has a Km of 0.56 micron for ethylmercuric chloride, a Km of 7.7 micron for methylmercuric chloride, and two Km values of 0.24 micron and over 200 micron for phenylmercuric acetate. The hydrolase has an isoelectric point of 5.5, requires the presence of EDTA and a sulfhydryl compound for activity, has a Stokes radius of 24 A, and has a molecular weight of about 43,000 +/- 4,000.
D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000201:enzymology*; D006867:Hydrolases / Q000378:metabolism*; D007700:Kinetics; D008628:Mercury / Q000378:metabolism*; D008970:Molecular Weight; D009941:Organomercury Compounds / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D010957:Plasmids; D011487:Protein Conformation
D009941:Organomercury Compounds; D010088:Oxidoreductases; C021551:mercuric reductase; D006867:Hydrolases; D008628:Mercury