title: NmerA, the metal binding domain of mercuric ion reductase, removes Hg2+ from proteins, delivers it to the catalytic core, and protects cells under glutathione-depleted conditions.
abstract: The ligand binding and catalytic properties of heavy metal ions have led to the evolution of metal ion-specific pathways for control of their intracellular trafficking and/or elimination. Small MW proteins/domains containing a GMTCXXC metal binding motif in a betaalphabetabetaalphabeta fold are common among proteins controlling the mobility of soft metal ions such as Cu(1+), Zn(2+), and Hg(2+), and the functions of several have been established. In bacterial mercuric ion reductases (MerA), which catalyze reduction of Hg(2+) to Hg(0) as a means of detoxification, one or two repeats of sequences with this fold are highly conserved as N-terminal domains (NmerA) of uncertain function. To simplify functional analysis of NmerA, we cloned and expressed the domain and catalytic core of Tn501 MerA as separate proteins. In this paper, we show Tn501 NmerA to be a stable, soluble protein that binds 1 Hg(2+)/domain and delivers it to the catalytic core at kinetically competent rates. Comparison of steady-state data for full-length versus catalytic core MerA using Hg(glutathione)(2) or Hg(thioredoxin) as substrate demonstrates that the NmerA domain does participate in acquisition and delivery of Hg(2+) to the catalytic core during the reduction catalyzed by full-length MerA, particularly when Hg(2+) is bound to a protein. Finally, comparison of growth curves for glutathione-depleted Escherichia coli expressing either catalytic core, full-length, or a combination of core plus NmerA shows an increased protection of cells against Hg(2+) in the media when NmerA is present, providing the first evidence of a functional role for this highly conserved domain.
authors.affiliation: Department of Pharmaceutical Chemistry, University of California-San Francisco, 600 16th Street, San Francisco, California 94143-2280, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Ledwidge
authors.forename: Bijal
authors.initials: B
authors.lastname: Patel
authors.forename: Aiping
authors.initials: A
authors.lastname: Dong
authors.forename: David
authors.initials: D
authors.lastname: Fiedler
authors.forename: Mat
authors.initials: M
authors.lastname: Falkowski
authors.forename: Jane
authors.initials: J
authors.lastname: Zelikova
authors.forename: Anne O
authors.initials: AO
authors.lastname: Summers
authors.forename: Emil F
authors.initials: EF
authors.lastname: Pai
authors.forename: Susan M
authors.initials: SM
authors.lastname: Miller
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers; D005978:Glutathione / Q000172:deficiency*; D005980:Glutathione Reductase; D006801:Humans; D008628:Mercury / Q000378:metabolism*; D008667:Metalloproteins / Q000378:metabolism*; D008958:Models, Molecular; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
chemical_list: D002413:Cations, Divalent; D017931:DNA Primers; D008667:Metalloproteins; D011994:Recombinant Proteins; D010088:Oxidoreductases; C021551:mercuric reductase; D005980:Glutathione Reductase; D008628:Mercury; D005978:Glutathione