The phosphorylation site of the Kdp-ATPase of Escherichia coli: site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit.
The potassium-translocating Kdp-ATPase of Escherichia coli shares common functional properties with eukaryotic P-type ATPases. The KdpB subunit has been identified as the catalytic subunit forming the phosphorylated intermediate. Substitution of Asp-307 in KdpB by Glu, Asn, Gln, Tyr, His, Ala or Ser by site-directed mutagenesis and the subsequent transfer of the point mutations to the chromosome revealed that the mutants were not functioning with respect to cell growth at low K+ concentrations and ATPase activity as well as phosphorylation capacity of the purified Kdp complex. These findings indicate that Asp-307 in KdpB is the phosphorylation site of the Kdp-ATPase. In contrast, replacement of the close but non-conserved Asp-300 by Asn or Glu has no immediate influence on the enzyme functions tested. However, the Km for K+ of the ATPase activity has been increased 30-fold compared with the wild-type enzyme.
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Universität Osnabrück, Fachbereich Biologie/Chemie, Arbeitsgruppe Mikrobiologie, Germany.
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Altendorf
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D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015139:Blotting, Southern; D027682:Cation Transport Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000254:growth & development; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011188:Potassium / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D029968:Escherichia coli Proteins; D010750:Phosphoproteins; D001224:Aspartic Acid; D000251:Adenosine Triphosphatases; C058166:potassium translocating Kdp-ATPase, E coli; D011188:Potassium