The role of heavy-metal ATPases,HMAs, in zinc and cadmium transport in rice

The P_1B-type heavy metal ATPases (HMAs) are diverse in terms of tissue distribution, subcellular localization, and metal specificity. Functional studies of HMAs have shown that these transporters can be divided into two subgroups based on their metal-substrate specificity: a copper (Cu)/silver (Ag) group and a zinc (Zn)/cobalt (Co)/cadmium (Cd)/lead (Pb) group. Studies on Arabidopsis thaliana and metal hyperaccumulator plants indicate that HMAs play an important role in the translocation or detoxification of Zn and Cd in plants. Rice possesses nine HMA genes, of which OsHMA1–OsHMA3 belong to the Zn/Co/Cd/Pb subgroup. OsHMA2 plays an important role in root-to-shoot translocation of Zn and Cd, and participates in Zn and Cd transport to developing seeds in rice. OsHMA3 transports Cd and plays a role in the sequestration of Cd into vacuoles in root cells. Modification of the expression of these genes might be an effective approach for reducing the Cd concentration in rice grains.     

Bacterial transition metal P(1B)-ATPases: transport mechanism and roles in virulence

P(1B)-type ATPases are polytopic membrane proteins that couple the hydrolysis of ATP to the efflux of cytoplasmic transition metals. This paper reviews recent progress in our understanding of the structure and function of these proteins in bacteria. These are members of the P-type superfamily of transport ATPases. Cu(+)-ATPases are the most frequently observed and best-characterized members of this group of transporters. However, bacterial genomes show diverse arrays of P(1B)-type ATPases with a range of substrates (Cu(+), Zn(2+), Co(2+)). Furthermore, because of the structural similarities among transitions metals, these proteins can also transport nonphysiological substrates (Cd(2+), Pb(2+), Au(+), Ag(+)). P(1B)-type ATPases have six or eight transmembrane segments (TM) with metal coordinating amino acids in three core TMs flanking the cytoplasmic domain responsible for ATP binding and hydrolysis. In addition, regulatory cytoplasmic metal binding domains are present in most P(1B)-type ATPases. Central to the transport mechanism is the binding of the uncomplexed metal to these proteins when cytoplasmic substrates are bound to chaperone and chelating molecules. Metal binding to regulatory sites is through a reversible metal exchange among chaperones and cytoplasmic metal binding domains. In contrast, the chaperone-mediated metal delivery to transport sites appears as a largely irreversible event. P(1B)-ATPases have two overarching physiological functions: to maintain cytoplasmic metal levels and to provide metals for the periplasmic assembly of metalloproteins. Recent studies have shown that both roles are critical for bacterial virulence, since P(1B)-ATPases appear key to overcome high phagosomal metal levels and are required for the assembly of periplasmic and secreted metalloproteins that are essential for survival in extreme oxidant environments.     

Biochemical characterization of AtHMA6/PAA1, a chloroplast envelope Cu(I)-ATPase

Copper is an essential plant micronutrient playing key roles in cellular processes, among them photosynthesis. In Arabidopsis thaliana, copper delivery to chloroplasts, mainly studied by genetic approaches, is thought to involve two P(IB)-type ATPases: AtHMA1 and AtHMA6/PAA1. The lack of biochemical characterization of AtHMA1 and PAA1, and more generally of plant P(IB)-type ATPases, is due to the difficulty of getting high amounts of these membrane proteins in an active form, either from their native environment or after expression in heterologous systems. In this study, we report the first biochemical characterization of PAA1, a plant copper-transporting ATPase. PAA1 produced in Lactococcus lactis is active, forming an aspartyl phosphate intermediate in the presence of ATP and the adequate metal ion. PAA1 can also be phosphorylated using inorganic phosphate in the absence of transition metal. Both phosphorylation types allowed us to demonstrate that PAA1 is activated by monovalent copper ions (and to a lower extent by silver ions) with an apparent affinity in the micromolar range. In agreement with these biochemical data, we also demonstrate that when expressed in yeast, PAA1 induces increased sensitivities to copper and silver. These data provide the first enzymatic characterization of a P(IB-1)-type plant ATPase and clearly identify PAA1 as a high affinity Cu(I) transporter of the chloroplast envelope.     

Functional expression of AtHMA4, a P1B-type ATPase of the Zn/Co/Cd/Pb subclass

Mechanisms are required by all organisms to maintain the concentration of essential heavy metals (e.g. Zn and Cu) within physiological limits and to minimise the detrimental effects of non-essential heavy metals (e.g. Cd). Heavy-metal P-type ATPases (HMAs) are a subgroup of the P-type ATPase superfamily that may contribute to metal homeostasis in plants. We cloned and characterised a member of this family, AtHMA4, from Arabidopsis thaliana that clusters with the Zn/Co/Cd/Pb subclass of HMAs on phylogenetic analysis. Sequencing of the AtHMA4 cDNA showed that it contained the conserved motifs found in all P-type ATPases and also motifs that are characteristic of heavy-metal ATPases. Escherichia coli mutants defective in the HMAs, CopA and ZntA, were used in functional complementation studies. AtHMA4 was able to restore growth at high [Zn] in the zntA mutant but not at high [Cu] in the copA mutant, suggesting a role in zinc transport. Heterologous expression of AtHMA4 in Saccharomyces cerevisiae made the yeast more resistant to Cd but did not affect sensitivity to other metals compared with vector-transformed controls. The organ specificity of AtHMA4 was analysed in Arabidopsis and showed that AtHMA4 was expressed in a range of tissues with highest expression in roots. AtHMA4 was upregulated in roots exposed to elevated levels of Zn and Mn but downregulated by Cd. Possible physiological roles of this transporter in Arabidopsis are discussed.     

Expression and mutagenesis of ZntA, a zinc-transporting P-type ATPase from Escherichia coli.

Cation-transporting P-type ATPases comprise a major membrane protein family, the members of which are found in eukaryotes, eubacteria, and archaea. A phylogenetically old branch of the P-type ATPase family is involved in the transport of heavy-metal ions such as copper, silver, cadmium, and zinc. In humans, two homologous P-type ATPases transport copper. Mutations in the human proteins cause disorders of copper metabolism known as Wilson and Menkes diseases. E. coli possesses two genes for heavy-metal translocating P-type ATPases. We have constructed an expression system for one of them, ZntA, which encodes a 732 amino acid residue protein capable of transporting Zn(2+). A vanadate-sensitive, Zn(2+)-dependent ATPase activity is present in the membrane fraction of our expression strain. In addition to Zn(2+), the heavy-metal ions Cd(2+), Pb(2+), and Ag(+) activate the ATPase. Incubation of membranes from the expression strain with [gamma-(33)P]ATP in the presence of Zn(2+), Cd(2+), or Pb(2+) brings about phosphorylation of two membrane proteins with molecular masses of approximately 90 and 190 kDa, most likely representing the ZntA monomer and dimer, respectively. Although Cu(2+) can stimulate phosphorylation by [gamma-(33)P]ATP, it does not activate the ATPase. Cu(2+) also prevents the Zn(2+) activation of the ATPase when present in 2-fold excess over Zn(2+). Ag(+) and Cu(+) appear not to promote phosphorylation of the enzyme. To study the effects of Wilson disease mutations, we have constructed two site-directed mutants of ZntA, His475Gln and Glu470Ala, the human counterparts of which cause Wilson disease. Both mutants show a reduced metal ion stimulated ATPase activity (about 30-40% of the wild-type activity) and are phosphorylated much less efficiently by [gamma-(33)P]ATP than the wild type. In comparison to the wild type, the Glu470Ala mutant is phosphorylated more strongly by [(33)P]P(i), whereas the His475Gln mutant is phosphorylated more weakly. These results suggest that the mutation His475Gln affects the reaction with ATP and P(i) and stabilizes the enzyme in a dephosphorylated state. The Glu470Ala mutant seems to favor the E2 state. We conclude that His475 and Glu470 play important roles in the transport cycles of both the Wilson disease ATPase and ZntA.     

Identification of Thlaspi caerulescens genes that may be involved in heavy metal hyperaccumulation and tolerance. Characterization of a novel heavy metal transporting ATPase

Thlaspi caerulescens is a heavy metal hyperaccumulator plant species that is able to accumulate extremely high levels of zinc (Zn) and cadmium (Cd) in its shoots (30,000 microg g(-1) Zn and 10,000 microg g(-1) Cd), and has been the subject of intense research as a model plant to gain a better understanding of the mechanisms of heavy metal hyperaccumulation and tolerance and as a source of genes for developing plant species better suited for the phytoremediation of metal-contaminated soils. In this study, we report on the results of a yeast (Saccharomyces cerevisae) complementation screen aimed at identifying candidate heavy metal tolerance genes in T. caerulescens. A number of Thlaspi genes that conferred Cd tolerance to yeast were identified, including possible metal-binding ligands from the metallothionein gene family, and a P-type ATPase that is a member of the P1B subfamily of purported heavy metal-translocating ATPases. A detailed characterization of the Thlaspi heavy metal ATPase, TcHMA4, demonstrated that it mediates yeast metal tolerance via active efflux of a number of different heavy metals (Cd, Zn, lead [Pb], and copper [Cu]) out of the cell. However, in T. caerulescens, based on differences in tissue-specific and metal-responsive expression of this transporter compared with its homolog in Arabidopsis (Arabidopsis thaliana), we suggest that it may not be involved in metal tolerance. Instead, we hypothesize that it may play a role in xylem loading of metals and thus could be a key player in the hyperaccumulation phenotype expressed in T. caerulescens. Additionally, evidence is presented showing that the C terminus of the TcHMA4 protein, which contains numerous possible heavy metal-binding His and Cys repeats residues, participates in heavy metal binding. When partial peptides from this C-terminal domain were expressed in yeast, they conferred an extremely high level of Cd tolerance and Cd hyperaccumulation. The possibilities for enhancing the metal tolerance and phytoremediation potential of higher plants via expression of these metal-binding peptides are also discussed.     

Conserved aspartic acid 714 in transmembrane segment 8 of the ZntA subgroup of P1B-type ATPases is a metal-binding residue

ZntA from Escherichia coli is a member of the P1B-type ATPase family that confers resistance specifically to Pb2+, Zn2+, and Cd2 salts by active efflux across the cytoplasmic membrane. P1B-type ATPases are important for homeostasis of metal ions such as Cu+, Ag+, Pb2+, Zn2+, Cd2+ Cu2+, and Co2+, with different subgroups showing specificity for different metal ions. Sequence alignments of P1B-type ATPases show that ZntA and close homologues have a strictly conserved Asp714 in the eighth transmembrane domain that is not conserved in other subgroups of P1B-type ATPases. However, in the sarcoplasmic reticulum Ca2+-ATPase, a structurally characterized P-type ATPase, the residue corresponding to Asp714 is a metal-binding residue. Four site-specific mutants at Asp714, D714E, D714H, D714A, and D714P, were characterized. A comparison of their metal-binding affinity with that of wtZntA revealed that Asp714 is a ligand for the metal ion in the transmembrane site. Thus, Asp714 is one of the residues that determine metal ion specificity in ZntA homologues. All four substitutions at Asp714 in ZntA resulted in complete loss of in vivo resistance activity and complete or large reductions in ATPase activity, though D714E and D714H retained the ability to bind metal ions with high affinity at the transmembrane site. Thus, the ability to bind metal ions with high affinity did not correlate with high activity. The metal-binding affinity of the N-terminal site remained unchanged in all four mutants. The affinities of the two metal-binding sites in wtZntA determined in this study are similar to values reported previously for the individual sites in isolated ZntA fragments.     

Characterization of the PIB-Type ATPases present in Thermus thermophilus

P(IB)-type ATPases transport heavy metals (Cu(2+), Cu(+), Ag(+), Zn(2+), Cd(2+), Co(2+)) across biomembranes, playing a key role in homeostasis and in the mechanisms of biotolerance of these metals. Three genes coding for putative P(IB)-type ATPases are present in the genome of Thermus thermophilus (HB8 and HB27): the TTC1358, TTC1371, and TTC0354 genes; these genes are annotated, respectively, as two copper transporter (CopA and CopB) genes and a zinc-cadmium transporter (Zn(2+)/Cd(2+)-ATPase) gene. We cloned and expressed the three proteins with 8His tags using a T. thermophilus expression system. After purification, each of the proteins was shown to have phosphodiesterase activity at 65°C with ATP and p-nitrophenyl phosphate (pNPP) as substrates. CopA was found to have greater activity in the presence of Cu(+), while CopB was found to have greater activity in the presence of Cu(2+). The putative Zn(2+)/Cd(2+)-ATPase was truncated at the N terminus and was, surprisingly, activated in vitro by copper but not by zinc or cadmium. When expressed in Escherichia coli, however, the putative Zn(2+)/Cd(2+)-ATPase could be isolated as a full-length protein and the ATPase activity was increased by the addition of Zn(2+) and Cd(2+) as well as by Cu(+). Mutant strains in which each of the three P-type ATPases was deleted singly were constructed. In each case, the deletion increased the sensitivity of the strain to growth in the presence of copper in the medium, indicating that each of the three can pump copper out of the cells and play a role in copper detoxification.     

Structural basis for metal binding specificity: the N-terminal cadmium binding domain of the P1-type ATPase CadA

In bacteria, P1-type ATPases are responsible for resistance to di- and monovalent toxic heavy metals by taking them out of the cell. These ATPases have a cytoplasmic N terminus comprising metal binding domains defined by a betaalphabetabetaalphabeta fold and a CXXC metal binding motif. To check how the structural properties of the metal binding site in the N terminus can influence the metal specificity of the ATPase, the first structure of a Cd(II)-ATPase N terminus was determined by NMR and its coordination sphere was investigated by X-ray absorption spectroscopy. A novel metal binding environment was found, comprising the two conserved Cys residues of the metal binding motif and a Glu in loop 5. A bioinformatic search identifies an ensemble of highly homologous sequences presumably with the same function. Another group of highly homologous sequences is found which can be referred to as zinc-detoxifying P1-type ATPases with the metal binding pattern DCXXC in the N terminus. Because no carboxylate groups participate in Cu(I) or Ag(I) binding sites, we suggest that the acidic residue plays a key role in the coordination properties of divalent cations, hence conferring a function to the N terminus in the metal specificity of the ATPase.     

The plant P1B-type ATPase AtHMA4 transports Zn and Cd and plays a role in detoxification of transition metals supplied at elevated levels

The transition metal Zn is essential for many physiological processes in plants, yet at elevated concentrations this, and the related non-essential metal Cd, can be toxic. Arabidopsis thaliana HMA4, belonging to the Type P1B subfamily of P-type ATPases, has recently been implicated in Zn nutrition, having a role in root to shoot Zn translocation. Using Arabidopsis insertional mutants, it is shown here that disruption of AtHMA4 function also results in increased sensitivity to elevated levels of Cd and Zn, suggesting that AtHMA4 serves an important role in metal detoxification at higher metal concentrations. AtHMA4 and a truncated form lacking the last 457 amino acids both confer Cd and Zn resistance to yeast but a mutant version of the full-length AtHMA4 (AtHMA4-C357G) does not; this demonstrates that the C-terminal region is not essential for this function. Evidence is presented that AtHMA4 functions as an efflux pump.     

The cysteine-rich amino-terminal domain of ZntA, a Pb(II)/Zn(II)/Cd(II)-translocating ATPase from Escherichia coli, is not essential for its function.

Soft metal-translocating P1-type ATPases have a distinctive amino-terminal domain that contains one to six copies of the conserved metal-binding motif, GXXCXXC. ZntA from Escherichia coli, a Pb(II)-, Zn(II)-, and Cd(II)-transporting ATPase, has an approximately 120 residue amino-terminal domain with one copy of the GXXCXXC motif as well as four additional cysteine residues. The function of this domain was investigated by constructing a mutant of ZntA lacking the first approximately 100 residues. The mutant, DeltaN-ZntA, was able to confer resistance to Pb(II), Zn(II), and Cd(II) salts, in a manner similar to ZntA. The soft metal dependent ATP hydrolysis activity of purified DeltaN-ZntA was characterized. Purified DeltaN-ZntA and ZntA were both inactivated by oxidation. The K(m) for MgATP was unchanged for DeltaN-ZntA relative to ZntA. DeltaN-ZntA displayed the same metal ion specificity as ZntA. Thiolates increased the activities of both ZntA and DeltaN-ZntA. The V(max) values for DeltaN-ZntA were approximately 3-fold lower than for ZntA for all three metal ions. Thus, the amino-terminal domain is not essential for the function of ZntA or for conferring specificity toward particular soft metals. Its function may be to increase the overall catalytic rate by increasing the rate of metal ion binding to the transporter. Residues involved in the ATP-dependent soft metal ion-translocating mechanism as well as those responsible for recognition of specific metal ions must be part of the core structure of the P1-type ATPases.     

Role of ENA ATPase in Na<Superscript>+</Superscript> efflux at high pH in bryophytes

Potassium or Na⁺ efflux ATPases, ENA ATPases, are present in all fungi and play a central role in Na⁺ efflux and Na⁺ tolerance. Flowering plants lack ENA ATPases but two ENA ATPases have been identified in the moss Physcomitrella patens, PpENA1 and PpENA2. PpENA1 mediates Na⁺ efflux in Saccharomyces cerevisiae. To propose a general function of ENA ATPases in bryophytes it was necessary to demonstrate that these ATPases mediate Na⁺ efflux in planta and that they exist in more bryophytes than P. patens. For these demonstrations (1) we cloned a third ATPase from P. patens, PpENA3, and studied the expression pattern of the three PpENA genes; (2) we constructed and studied the single and double Δppena1 and Δppena2 mutants; and (3) we cloned two ENA ATPases from the liverwort Marchantia polymorpha, MpENA1 and MpENA2, and expressed them in S. cerevisiae. The results from the first two approaches revealed that the expression of ENA ATPases was greatly enhanced at high pH and that Na⁺ efflux at high pH depended on PpENA1. The ENA1 ATPase of M. polymorpha suppressed the defective growth of a S. cerevisiae mutant at high K⁺ or Na⁺ concentrations, especially at high K⁺.     

Functional analysis of chimeric proteins of the Wilson Cu(I)-ATPase (ATP7B) and ZntA, a Pb(II)/Zn(II)/Cd(II)-ATPase from Escherichia coli

ATP7B, the Wilson disease-associated Cu(I)-transporter, and ZntA from Escherichia coli are soft metal P1-type ATPases with mutually exclusive metal ion substrates. P1-type ATPases have a distinctive amino-terminal domain containing the conserved metal-binding motif GXXCXXC. ZntA has one copy of this motif while ATP7B has six copies. The effect of interchanging the amino-terminal domains of ATP7B and ZntA was investigated. Chimeric proteins were constructed in which either the entire amino-terminal domain of ATP7B or only its sixth metal-binding motif replaced the amino-terminal domain of ZntA. Both chimeras conferred resistance to lead, zinc, and cadmium salts but not to copper salts. The purified chimeras displayed activity with lead, cadmium, zinc, and mercury, which are substrates of ZntA. There was no activity with copper or silver, which are substrates of ATP7B. The chimeras were 2-3-fold less active than ZntA. Thus, the amino-terminal domain of P1-type ATPases cannot alter the metal specificity determined by the transmembrane segment. Also, these results suggest that this domain interacts with the rest of the transporter in a metal ion-specific manner; the amino-terminal domain of ATP7B cannot replace that of ZntA in restoring full catalytic activity.     

Expression of ZntA, a zinc-transporting P1-type ATPase, is specifically regulated by zinc and cadmium

All cells have developed various mechanisms to regulate precisely the availability of important micronutrients such as zinc and copper; in many cells, this regulation is mediated by P1-type ATPases. Most of the P1-ATPases have been described very recently, and little is known about their molecular mechanism and regulation. Here, we demonstrate that the expression of ZntA, a Zn,Cd-transporting P-type ATPase of Escherichia coli, is specifically regulated by the transported cations, cadmium and zinc. Nickel, cobalt, and copper did not induce the expression of ZntA, even when present at concentrations as high as 0.6-1 mM. The effect of zinc and cadmium on the ZntA expression is concentration dependent, the apparent Km for Cd (19 microM) being markedly lower than that for Zn (100 microM). This metal selectivity is opposite to the known metal selectivity of transport by ZntA. Thus, we speculate that, to maintain zinc concentrations in the cell in the presence of cadmium, ZntA probably interacts with other proteins that modulate the ZntA selectivity towards transported cations.     

New findings on evolution of metal homeostasis genes: evidence from comparative genome analysis of bacteria and archaea

In order to examine the natural history of metal homeostasis genes in prokaryotes, open reading frames with homology to characterized P(IB)-type ATPases from the genomes of 188 bacteria and 22 archaea were investigated. Major findings were as follows. First, a high diversity in N-terminal metal binding motifs was observed. These motifs were distributed throughout bacterial and archaeal lineages, suggesting multiple loss and acquisition events. Second, the CopA locus separated into two distinct phylogenetic clusters, CopA1, which contained ATPases with documented Cu(I) influx activity, and CopA2, which contained both efflux and influx transporters and spanned the entire diversity of the bacterial domain, suggesting that CopA2 is the ancestral locus. Finally, phylogentic incongruences between 16S rRNA and P(IB)-type ATPase gene trees identified at least 14 instances of lateral gene transfer (LGT) that had occurred among diverse microbes. Results from bootstrapped supported nodes indicated that (i) a majority of the transfers occurred among proteobacteria, most likely due to the phylogenetic relatedness of these organisms, and (ii) gram-positive bacteria with low moles percent G+C were often involved in instances of LGT. These results, together with our earlier work on the occurrence of LGT in subsurface bacteria (J. M. Coombs and T. Barkay, Appl. Environ. Microbiol. 70:1698-1707, 2004), indicate that LGT has had a minor role in the evolution of P(IB)-type ATPases, unlike other genes that specify survival in metal-stressed environments. This study demonstrates how examination of a specific locus across microbial genomes can contribute to the understanding of phenotypes that are critical to the interactions of microbes with their environment.     

Metal-binding characteristics of the amino-terminal domain of ZntA: binding of lead is different compared to cadmium and zinc

ZntA from Escherichia coli, a P1-type ATPase, specifically transports Pb(II), Zn(II), and Cd(II). Most P1-type ATPases have an N-terminal domain that contains one or more copies of the conserved metal-binding motif, GXXCXXC. In ZntA, the N-terminal domain has approximately 120 residues with a single GXXCXXC motif, as well as four additional cysteine residues as part of the CCCDGAC motif. The metal-binding specificity and affinity of this domain in ZntA was investigated. Isolated proteins, N1-ZntA and N2-ZntA, containing residues 1-111 and 47-111 of ZntA, respectively, were characterized. N1-ZntA has both the CCCDGAC and GXXCXXC motifs, while N2-ZntA has only the GXXCXXC motif. ICP-MS measurements showed that N1-ZntA can bind both divalent metal ions such as Cd(II), Pb(II), and Zn(II) and monovalent metal ions such as Ag(I), with a stoichiometry of 1. N2-ZntA can bind Zn(II) and Cd(II) with a stoichiometry of 1 but not Pb(II). The affinity of N1-ZntA for Zn(II), Pb(II), and Cd(II) was measured by competition titration with metallochromic indicators. Association constants of approximately 10(8) M(-)(1) were obtained for Zn(II), Pb(II), and Cd(II) binding to N1-ZntA. To investigate whether the CCCDGAC sequence has an important role in binding specifically Pb(II), a mutant of ZntA, which lacked the first 46 residues, was constructed. This mutant, Delta46-ZntA, had the same activity as wtZntA with respect to Cd(II) and Zn(II). However, its activity with Pb(II) was similar to the mutant DeltaN-ZntA, which lacks the entire N-terminal domain (Mitra, B., and Sharma, R. (2001) Biochemistry 40, 7694-7699). Thus, binding of Pb(II) appears to involve different ligands, and possibly geometry, compared to Cd(II) and Zn(II).     

Novel Zn2+ coordination by the regulatory N-terminus metal binding domain of Arabidopsis thaliana Zn(2+)-ATPase HMA2

Arabidopsis thaliana HMA2 is a Zn2+ transporting P1B-type ATPase required for maintaining plant metal homeostasis. HMA2 and all eukaryote Zn2+-ATPases have unique conserved N- and C-terminal sequences that differentiate them from other P1B-type ATPases. Homology modeling and structural comparison by circular dichroism indicate that the 75 amino acid long HMA2 N-terminus shares the betaalphabetabetaalpha folding present in most P1B-type ATPase N-terminal metal binding domains (N-MBDs). However, the characteristic metal binding sequence CysXXCys is replaced by Cys17CysXXGlu21, a sequence present in all plant Zn2+-ATPases. The isolated HMA2 N-MBD fragment binds a single Zn2+ (Kd 0.18 microM), Cd2+ (Kd 0.27 microM), or, with less affinity, Cu+ (Kd 13 microM). Mutagenesis studies indicate that Cys17, Cys18, and Glu21 participate in Zn2+ and Cd2+ coordination, while Cys17 and Glu21, but not Cys18, are required for Cu+ binding. Interestingly, the Glu21Cys mutation that generates a CysCysXXCys site is unable to bind Zn2+ or Cd2+ but it binds Cu+ with affinity (Kd 1 microM) higher than wild type N-MBD. Truncated HMA2 lacking the N-MBD showed reduced ATPase activity without significant changes in metal binding to transmembrane metal binding sites. Likewise, ATPase activity of HMA2 carrying mutations Cys17Ala, Cys18Ala, and Glu21Ala/Cys was also reduced but showed a metal dependence similar to the wild type enzyme. These observations suggest that plant Zn2+-ATPase N-MBDs have a folding and function similar to Cu+-ATPase N-MBDs. However, the unique Zn2+ coordination via two thiols and a carboxyl group provides selective binding of the activating metals to these regulatory domains. Metal binding through these side chains, although found in different sequences, appears as a common feature of both bacterial and eukaryotic Zn2+-ATPase N-MBDs.     

The metal specificity and selectivity of ZntA from Escherichia coli using the acylphosphate intermediate

ZntA from Escherichia coli is a P-type ATPase that confers resistance to Pb(II), Zn(II), and Cd(II) in vivo. We had previously shown that purified ZntA shows ATP hydrolysis activity with the metal ions Pb(II), Zn(II), and Cd(II). In this study, we utilized the acylphosphate formation activity of ZntA to further investigate the substrate specificity of ZntA. The site of phosphorylation was Asp-436, as expected from sequence alignments. We show that in addition to Pb(II), Zn(II), and Cd(II), ZntA is active with Ni(II), Co(II), and Cu(II), but not with Cu(I) and Ag(I). Thus, ZntA is specific for a broad range of divalent soft metal ions. The activities with Ni(II), Co(II), and Cu(II) are extremely low; the activities with these non-physiological substrates are 10-20-fold lower compared with the values obtained with Pb(II), Zn(II), and Cd(II). Similar results were obtained with DeltaN-ZntA, a ZntA derivative lacking the amino-terminal metal binding domain. By characterizing the acylphosphate formation reaction in ZntA in detail, we show that a step prior to enzyme phosphorylation, most likely the metal ion binding step, is the slow step in the reaction mechanism in ZntA. The low activities with Ni(II), Co(II), and Cu(II) are because of a further decrease in the rate of binding of these metal ions. Thus, metal ion selectivity in ZntA and possibly other P1-type ATPases is based on the charge and the ligand preference of particular metal ions but not on their size.