C-terminal cysteines of Tn501 mercuric ion reductase.
Mercuric ion reductase (MerA) catalyzes the reduction of Hg(II) to Hg(0) as the last step in the bacterial mercury detoxification pathway. A member of the flavin disulfide oxidoreductase family, MerA contains an FAD prosthetic group and redox-active disulfide in its active site. However, the presence of these two moieties is not sufficient for catalytic Hg(II) reduction, as other enzyme family members are potently inhibited by mercurials. We have previously identified a second pair of active site cysteines (Cys558 Cys559 in the Tn501 enzyme) unique to MerA, that are essential for high levels of mercuric ion reductase activity [Moore, M. J., & Walsh, C. T. (1989) Biochemistry 28, 1183; Miller, S. M., et al. (1989) Biochemistry 28, 1194]. In this paper, we have examined the individual roles of Cys558 and Cys559 by site-directed mutagenesis of each to alanine. Phenotypic analysis indicates that both merA mutations result in a total disruption of the Hg(II) detoxification pathway in vivo, while characterization of the purified mutant enzymes in vitro shows each to have differential effects on catalytic function. Compared to wild-type enzyme, the C558A mutant shows a 20-fold reduction in kcat and a 10-fold increase in Km, for an overall decrease in catalytic efficiency of 200-fold in kcat/Km. In contrast, mutation of Cys559 to alanine results in less than a 2-fold reduction in kcat and an increase in Km of only 4-5 fold for an overall decrease in catalytic efficiency of only ca. 10-fold in vitro. From these results, it appears that Cys558 plays a more important role in forming the reducible complex with Hg(II), while both Cys558 and Cys559 seem to be involved in efficient scavenging (i.e., tight binding) of Hg(II).
Moore
M J
MJ
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Miller
S M
SM
Walsh
C T
CT
D000409:Alanine; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003545:Cysteine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008627:Mercuric Chloride / Q000494:pharmacology; D008628:Mercury / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009536:Niacinamide / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D013053:Spectrophotometry; D013329:Structure-Activity Relationship; D013438:Sulfhydryl Compounds / Q000032:analysis; D014169:Transformation, Bacterial
D013438:Sulfhydryl Compounds; D009536:Niacinamide; D008627:Mercuric Chloride; D010088:Oxidoreductases; C021551:mercuric reductase; D008628:Mercury; D003545:Cysteine; D000409:Alanine