Heavy Metal P-type ATPases

Acylphosphate formation is characteristic for P-type ATPases and involves the transfer of the y-phosphate of ATP to an aspartic acid residue to form a high-energy enzyme intermediate. The phosphorylated aspartic acid residue is located in the sequence DKTGT, which is universally conserved in all members of the P-type superfamily. By this criterion, CopA and CopB of En. hirae are clearly members of the P-type superfamily of ATPases and probably function by the same underlying mechanism. Vanadate sensitivity is another hallmark of P-type ATPases. CopA and CopB were inhibited by vanadate with /50 values of around 0.1 mM. This is a low vanadate sensitivity compared to /50 values in the micromolar to submicromolar range observed for non-heavy metal P-type ATPases. 

Fig. 1. General structure of (A) non-heavy metal P-type ATPase and (B) heavy metal P-type ATPase. Metal-binding sites are putative copper-binding motifs GMxCxxC phosphatase domain, a cytosolic loop with a conserved region(s), e.g., TGEA, essential for dephosphorylation of the transient acyl phosphate P-DKTG is the...

One of the characteristic motifs of heavy metal P-type ATPases is the CPX (commonly CPC) motif within transmembrane domain 6, widely regarded as a part of the cation channel (Silver and Phung, 1996) (Fig. 1). The possible role for these cysteines in the transmembrane domain may be the coordination of a heavy metal during its translocation through the cation channel. While these cysteines have proved to be essential for the function of some HMPAs, their exact role in catalysis is yet to be fully understood (Bissig et al., 2001 Forbes and Cox, 2000). 

Another common feature of heavy metal P-type ATPases, which has attracted a great deal of interest, is the presence of at least one putative MBS, commonly GMxCxxC, at the N-terminus of these proteins. Interestingly, the number of MBSs increases from 1 or 2 in bacteria and yeast to 3 in Caenorhabditis elegans and 5 or 6 in mammalian CuPAs (Fig. 1). This increase was probably due to the amplification of a portion of coding sequence, as MBSs are highly conserved between various heavy metal transporters, including the conserved leucine residue in the position +8 or +11, isoleucine in the position +8, and phenylalanine in the position... 

P-type ATPases. This is an unexpected finding as generally the cation specihcity of P-type ATPases is restricted to the amino acid composition of cation channels in transmemhrane domains (Axelsen and Palmgren, 1998 Mpller et al., 1996). It is probable that other, as yet to be identihed, intramolecular interactions may be important for the specihc cation transport by heavy metal P-type ATPases. Cysteine residues in transmembrane domain 6, which form a characteristic CPx (commonly CPC) motif, are commonly regarded as core elements of the cation channel in CuPAs. However, there is no direct evidence to date to support that assumption. 

Zinc efflux is mediated by a zinc exporter known as ZntA (Zn + transport or tolerance), a membrane protein which was identified through studies of bacterial strains that were hypersensitive to zinc and cadmium. Sequence inspection revealed that ZntA was a member of the family of cation transport P-type ATPases, a major family of ion-translocating membrane proteins in which ATPase activity in one portion of the protein is used to phophorylate an aspartate within a highly conserved amino acid sequence, DKTG, in another portion of the protein. The cysteine rich N-terminus of these soft metal transport proteins contains several metal-binding sites. How the chemical energy released by ATP hydrolysis results in metal ion transport is not yet known, in part because there is only partial information about the structures of these proteins. The bacterial zinc exporter also pumps cadmium and lead and is therefore also involved in protection from heavy metal toxicity (see Metal Ion Toxicity). 

A selected group of genes known to be inner membrane transporters, such as those for the uptake of substrates, synthesis of ATP, pH regulation such as NaVH" exchange, and heavy metal concentration such as the P-type ATPases. Many of these could be potential targets for elimination of the infection. 

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