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

Abstract

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 ∼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 ∼100 residues. The mutant, ΔN-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 ΔN-ZntA was characterized. Purified ΔN-ZntA and ZntA were both inactivated by oxidation. The K_m for MgATP was unchanged for ΔN-ZntA relative to ZntA. ΔN-ZntA displayed the same metal ion specificity as ZntA. Thiolates increased the activities of both ZntA and ΔN-ZntA. The V_max values for ΔN-ZntA were ∼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.