The ATP Hydrolytic Activity of Purified ZntA, a Pb(II)/Cd(II)/Zn(II)-translocating ATPase from Escherichia coli

Abstract

ZntA, a soft metal-translocating P1-type ATPase from Escherichia coli, confers resistance to Pb(II), Cd(II), and Zn(II). ZntA was expressed as a histidyl-tagged protein, solubilized from membranes with Triton X-100, and purified to homogeneity. The soft metal-dependent ATP hydrolysis activity of purified ZntA was characterized. The activity was specific for Pb(II), Cd(II), Zn(II), and Hg(II), with the highest activity obtained when the metals were present as thiolate complexes of cysteine or glutathione. The maximal ATPase activity of ZntA was ϳ3 mol/(mg⅐min) obtained with the Pb(II)-thiolate complex. In the absence of thiolates, Cd(II) inhibits ZntA above pH 6, whereas the Cd(II)-thiolate complexes stimulate activity, suggesting that a metal-thiolate complex is the true substrate in vivo. These results are consistent with the physiological role of ZntA as mediator of resistance to toxic concentrations of the divalent soft metals, Pb(II), Cd(II), and Zn(II), by ATP-dependent efflux. Our results confirm that ZntA is the first Pb(II)-dependent ATPase discovered to date.

A P-type ATPase importer that discriminates between essential and toxic transition metals

“…Due to the chemical similarities between Cu ϩ and Ag ϩ and between Zn 2ϩ and Cd 2ϩ , it is not surprising that P-type exporters specialize in detoxifying one of these two subsets (7,13,14,21,23,24). We have determined the metal specificities for 5 previously uncharacterized P-type transition-metal exporters, along with tentative assignments for 3 additional pumps exhibiting low levels of activity.…”

“…This notion is further supported by functional analysis of various P-type pumps harboring these amino acid motifs. In characterized systems (13,14), pumps harboring amino acid sequences specific to monovalent metals indeed transported only copper and silver, whereas those harboring amino acid sequences specific to divalent metals transported only zinc, cadmium, and lead.To further our understanding of substrate recognition by transition-metal pumps, we have cloned and expressed 18 uncharacterized prokaryotic P-type ATPases (15). Initially, we tested the abilities of the expressed proteins to restore tolerance to external concentrations of copper, silver, zinc, and cadmium in metalsensitive Escherichia coli strains (16).…”

“…This notion is further supported by functional analysis of various P-type pumps harboring these amino acid motifs. In characterized systems (13,14), pumps harboring amino acid sequences specific to monovalent metals indeed transported only copper and silver, whereas those harboring amino acid sequences specific to divalent metals transported only zinc, cadmium, and lead.…”

Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis

“…Cd has been demonstrated to inactivate or denature proteins by binding to sulfhydryl groups, which causes cellular damages by displacing cofactors from a variety of proteins, including transcription factors and enzymes (Goyer, 1997;Sharma et al, 2000;Schützendübel et al, 2001;Lin and Aarts, 2012). Moreover, Cd also induces oxidative stress, which in turn mediates cellular damage in various plants and animals (Shim et al, 2009;Gallego et al, 2012).…”

“…In addition, a lot of key genes have been shown to be involved in Cd detoxification and tolerance in plants. These include ZNT1, OsHMA9, OsNRAMP5, ZntA, CAD2, AtPDR12, AtPDR8, AtATM3, ACBP1, Nramp5, EIN2, and MAN3 (Pence et al, 2000;Sharma et al, 2000;Shiraishi et al, 2000;Lee et al, 2003Lee et al, , 2007Kim et al, 2006Kim et al, , 2007Xiao et al, 2008;Cao et al, 2009;Lin and Aarts, 2012;Sasaki et al, 2012;Ishikawa et al, 2012;Chen et al, 2015). It was also found that Heat shock transcription factor A4a (HsfA4a) confers Cd tolerance by up-regulating MT gene expression in wheat (Triticum aestivum) and rice (Oryza sativa; Shim et al, 2009).…”