Membrane topology of CadA cadmium resistant ATPase in Staphylococcus aureus

• Main Authors: Yung-Feng Lin (林詠峰), 林詠
• Other Authors: Kan-Jen Tsai
• Format: Others
• Language: en_US
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/mw3cc6

碩士 === 中山醫學院 === 醫學研究所 === 89 === Bacterial metal resistances evolved originally from the exposure of natural sources, and become more severe in recent years after the usages of therapeutic drugs. Some of these bacterial resistant mechanisms are raised from additive mutations of bacterial genes. Previously, the staphylococcal resistance to cadmium carried by pI258 penicillinase plasmid has been extensively studied. Biochemical characterization of the resistance has revealed that a CadA Cd2+-ATPase mediates the resistance phenomena and this protein has been categorized into a P-type ATPase enzyme family. Due to their difference in certain structural features, CadA and other P-type ATPases for transporting heavy metal ions have been grouped into a new enzyme family, called CPx-type ATPases. In this proposed study, we have investigated the CadA topography and, as a model, to characterize those functional domains unique for the CPx-ATPases. Using the phoA (alkaline phosphatase) and lacZ (β-galactosidase) gene fusion methods, a series of 22 cadA-phoA and 22 cadA-lacZ fusions were created throughout the cadA gene. Based on the reporter enzyme activities measured in this study, we have determined that there are eight transmembrane segments (TMs) in CadA, although the third and forth segments might not traverse the membrane completely. Both the N-terminus and the C-terminus of CadA protein are located within the cytoplasm. This topological model is similar to that of the homologous ATPase found in Helicobacter pylori except the description of the TM3 and TM4. Besides, some significant differences have been found between these two pumps, including a highly hydrophobic region just behind the putative ATP binding site found in staphylococcal CadA but not in the helicobacter version of CadA, and an asymmetric arrangement of charged residues between these two ATPases. Anyway, we proposed a more detailed structure model of this kind of ATPases using staphylococcal CadA as an example.