Heavy metal transport CPx-ATPases from the thermophile Archaeoglobus fulgidus.
PIB-type ATPases transport diverse heavy metals (Cu(+), Ag(+), Cu(2+). Zn(2+), Cd(2+), Pb(2+), Co(2+)) across membranes. Toward understanding their mechanisms of metal selectivity, we are studying thermophilic archaeal PIB-type ATPases. Like other PIB ATPases, these are characterized by the presence of a cation binding CPX sequence in their 6th transmembrane segment and by cytoplasmic N-terminus metal binding domains (N-MBDs). CopA and CopB from the thermophile Archaeoglobus fulgidus were cloned and expressed in E. coli. The resulting proteins were purified in a soluble active form. Typical yields were in the order of 3-5 mg of pure protein per liter of bacterial culture. Both enzymes showed maximum activity at 75-85 degrees C. CopA was activated by Ag(+)>Cu(+) while CopB was activated by Cu(2+)>Ag(+)>Cu(+). The differences in enzyme selectivity can be explained by different consensus sequences in the transmembrane cation binding domain (CopA: CPC, CopB: CPH). Mutagenesis studies show that the cysteines in the transmembrane CPC site of CopA are necessary for enzyme function, while those in the N-MBD (CXXC), although not essential, are required for maximum enzyme activity. Different from CopA, CopB has a His-rich N-MBD. Removal of this domain reduced enzyme activity without affecting enzyme selectivity. These studies show that these enzymes are an excellent system for structural functional studies directed to explain the mechanisms of metal selectivity by PIB ATPases.
Argüello
José M
JM
Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Massachusetts 01609, USA. arguello@wpi.edu
Mandal
Atin K
AK
Mana-Capelli
Sebastian
S
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D019611:Archaeoglobus fulgidus / Q000201:enzymology* / Q000235:genetics*; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D000079922:Copper Transport Proteins; D000073840:Copper-Transporting ATPases; D006358:Hot Temperature; D007700:Kinetics; D019216:Metals, Heavy / Q000493:pharmacokinetics*; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity
D027682:Cation Transport Proteins; D000079922:Copper Transport Proteins; D019216:Metals, Heavy; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; C088359:CopB ATPase, Enterococcus hirae; D000073840:Copper-Transporting ATPases