ChenChou Wu
Identification and characterization of the Cd2+ transport sites of CadA, the Cd2+-ATPase from Listeria monocytogenes
Published on 17 January 2005
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Thesis presented January 17, 2005
Abstract:
P1-type ATPases transport heavy metals across the membrane against their electrochemical gradient using the hydrolysis of ATP as energy source. In 1992, a study reported that 36% of
Listeria monocytogenes were resistant to high Cd2+ concentrations. This resistance was associated to the presence of plasmids, among which pLm74 presents an open reading frame coding for a polypeptide of 711 amino acids, named CadA. This polypeptide possesses 3 consensus sequences of P-type ATPases, the DKTGT, MXTGD and TGDGXNDXP motifs, as well as 2 consensus sequences of P1-type ATPases, the CXXC and CPC motifs. In the present work we expressed CadA in the yeast
Saccharomyces cerevisae and used the phenotype of sensitivity to cadmium induced by CadA as a screening tool for mutants produced by site-directed mutagenesis. We also demonstrated that CadA can use Cd-ATP instead of Mg-ATP as ATP substrate to accomplish its enzymatic cycle. Four transmembrane helices (3, 4, 6 and 8) might constitute the Cd2+ transport site of CadA. Among them M149, C354 and T684 might participate in transport sites whereas E164 and C356 could be important in the dissociation process of cadmium. P355 and D692 could be necessary for the phosphorylation. Finally, the study of chimeric ATPases suggests a regulatory role of the ATP binding domain.
Keywords:
CadA, type P1 ATPase, cadmium,
Listeria monocytogenes,
Saccharomyces cerevisiae, transport site
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