Homeostasis in Enterococcus hirae

CopA of En. hirae could be expressed in Escherichia coli and purified to homogeneity by Ni-NTA affinity chromatography by means of an added histidine tag (Wunderh-Ye and Solioz, in press). Purified CopA has a pH optimum of 6.3 and a for ATP of 0.2 mM. The enzyme forms an acylphosphate intermediate, which is a hallmark of P- and CPx-type ATPases (Pedersen and Carafoh, 1987b). Purified CopA can now serve in the analysis of mechanistic aspects of copper transport and in the characterization of structure-function relationships. 

CopB was shown to catalyze ATP-driven copper(I) and sUver(I) transport into native membrane vesicles of En. hirae. Since only inside-out oriented ATPase molecules were active in this transport assay, this corresponds to copper extrusion by CopB in vivo. Copper transport by vesicles took place only under reducing conditions. Cu(I) rather than Cu(II) was thus the transported species. Use of null mutants in copA, copB, orcoj Aand copB made it possible to attribute the observed transport to the activity of the CopB ATPase. Copper transport exhibited an apparent for Cu+ of 1 pM and a Umax of 0.07 nmol/min/mg of membrane protein. Ag+ was transported with a similar affinity and at a similar rate (Solioz and Oder-matt, 1995). Since Cu and Ag+ were complexed to Tris buffer and dithiothreitol present in the assay, the values must be considered as relative only. The results obtained with membrane vesicles were further supported by evidence of Ag+ extrusion from whole cells, preloaded with this isotope. Again, transport depended on the presence of functional 

and the corresponding knockout strains exhibited no silver extrusion (Odermattct a/., 1994). These hndings suggest that CopB functions as a Cu+/Ag+-ATPase for the export of Cu+ and Ag+ in vivo. 

Vanadate, a diagnostic inhibitor of P-type ATPases, showed an interesting biphasic pattern of inhibition of ATP-driven copper and silver transport by CopB maximal inhibition was observed at 40 pM VO4 for Cu+ transport and at 60 pM for Ag+ transport. At higher concentra- 

Regulation of Expression by Copper and Copper Chaperone Function 

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Figure 12.2 Copper chaperone function, (a) Copper homeostasis in Enterococcus hirae is affected by the proteins encoded by the cop operon. CopA, Cu1+-import ATPase CopB, Cu1+-export ATPase CopY, Cu1+-responsive repressor copZ, chaperone for Cu1+ delivery to CopY. (b) The CTR family of proteins transports copper into yeast cells. Atxlp delivers copper to the CPx-type ATPases located in the post Golgi apparatus for the maturation of Fet3p. (c) Coxl7p delivers copper to the mitochondrial intermembrane space for incorporation into cytochrome c oxidase (CCO). (d) hCTR, a human homologue of CTR, mediates copper-ion uptake into human cells. CCS delivers copper to cytoplasmic Cu/Zn superoxide dismutase (SOD1). Abbreviations IMM, inner mitochondrial membrane OMM, outer mitochondrial membrane PM, plasma membrane PGV, post Golgi vessel. Reprinted from Harrison et al., 2000. Copyright (2000), with permission from Elsevier Science.

Solioz, M. and Stoyanov, J.V. (2003) Copper homeostasis in Enterococcus hirae, FEMS Microbiol. Rev., 27, 183-195. 

Fig. 5. Copper homeostasis in Enterococcus hirae. Under copper-limiting conditions, copper is pumped into the cell by CopA. The CopZ copper chaperone picks up copper at this site of entry. Under physiological copper conditions, Zn(II)CopY binds to the promoter and represses transcription of the cop operon. Under conditions of copper excess, Cu-CopZ donates Cu(I) to CopY, which leads to the replacement of the Zn(II), loss of DNA-binding affinity, and ultimately synthesis of the operon products. Excess copper is secreted by the CopB efflux pump. The substrate for this pump may be a copper-glutathione (GSH) complex, rather than Cu-CopZ.

Odermatt, A, Suter, H., Krapt, R., and Solioz, M. (1993). Primary structure of two P-type ATPases involved in copper homeostasis in Enterococcus hirae. J. Biol. Chem. 268, 12775-12779. 

Because of their importance in many enzymes, bacteria have had to develop uptake systems for copper and zinc. Copper uptake (and homeostasis, which is discussed in Chapter 8) has been extensively studied in the Gram-positive bacterium Enterococcus hirae. Two of the genes in the cop operon, copA and copB, encode membrane ATPases. An extracellular reductase CorA reduces Cu2+ to Cu+, which is taken up by CopA when copper is limiting. 

Copper ion homeostasis in prokaryotes involves Cu ion efflux and sequestration. The proteins involved in these processes are regulated in their biosynthesis by the cellular Cu ion status. The best studied bacterial Cu metalloregulation system is found in the gram-positive bacterium Enterococcus hirae. Cellular Cu levels in this bacterium control the expression of two P-type ATPases critical for Cu homeostasis (Odermatt and Solioz, 1995). The CopA ATPase functions in Cu ion uptake, whereas the CopB ATPase is a Cu(I) efflux pump (Solioz and Odermatt, 1995). The biosynthesis of both ATPases is regulated by a Cu-responsive transcription factor, CopY (Harrison et al., 2000). In low ambient Cu levels Cop Y represses transcription of the two ATPase genes. On exposure to Cu(I), CopY dissociates from promoter/operator sites on DNA with a for Cu of 20 jlM (Strausak and Solioz, 1997). Transcription of copA and copB proceeds after dissociation of CuCopY. The only other metal ions that induce CopY dissociation from DNA in vitro are Ag(I) and Cd(II), although the in vivo activation of copA and copB is specihc to Cu salts. The CuCopY complex is dimeric with two Cu(I) ions binding per monomer (C. T. Dameron, personal communication). The structural basis for the Cu-induced dissociation of CopY is unknown. Curiously, CopY is also activated in Cu-dehcient cells, but the mechanism is distinct from the described Cu-induced dissociation from DNA (Wunderh-Ye and Solioz, 1999). 

In the gram-positive bacteria Enterococcus hirae, copper homeostasis is maintained by the cop operon, consisting of the copA, copB, copY, and copZ... 

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