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Heavy Metal Transport CPx‐ATPases from the Thermophile Archaeoglobus fulgidus
Author(s) -
ARGÜELLO JOSÉ M.,
MANDAL ATIN K.,
MANACAPELLI SEBASTIAN
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2003.tb07162.x
Subject(s) - thermophile , enzyme , transmembrane domain , biochemistry , atpase , chemistry , transmembrane protein , membrane , receptor
A bstract : 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°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.