Spermidine-preferential uptake system in Escherichia coli. ATP hydrolysis by PotA protein and its association with membrane.
PotA protein, one of the components of the spermidine-preferential uptake system in Escherichia coli, was purified to homogeneity, and some of its properties were examined. PotA protein showed Mg(2+)-and SH-dependent ATPase activity. The specific activity was approximately 400 nmol/min/mg of protein and the Km value for ATP was 385 microM. The nature of the ATP binding site was explored by identification of the amino acid residue photoaffinity-labeled with 8-azido-ATP. It was found that 8-azido-ATP was attached to cysteine 26. In the spermidine transport-deficient mutant E. coli NH1596, valine 135 of PotA protein, which is located between two consensus amino acid sequences for nucleotide binding (50-57 and 168-173), was replaced by methionine (Kashiwagi, K., Miyamoto, S., Nukui, E., Kobayashi, H., and Igarashi, K. (1993) J. Biol. Chem. 268, 19358-19363). This mutated PotA protein could be labeled with 8-azido-ATP, but showed very low ATPase activity. To identify which cysteine is involved in the function of potA protein, cysteines 26, 54, and 276 were replaced by alanine, threonine, and alanine, respectively. Among the three mutated PotA proteins, the mutated PotA protein C54T only lost both ATPase and spermidine uptake activities. The results taken together indicate that the adenine portion of ATP interacts with a domain close to the NH2-terminal end of PotA protein, and active centers of ATP hydrolysis are located both within and between the two consensus amino acid sequences for nucleotide binding. Association of PotA protein with membranes was strengthened by the existence of channel forming PotB and PotC proteins. ATPase of PotA protein was inhibited by spermidine, suggesting that uptake inhibition by spermidine may function during this process.
Kashiwagi
K
K
Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Endo
H
H
Kobayashi
H
H
Takio
K
K
Igarashi
K
K
D018528:ATP-Binding Cassette Transporters; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000345:Affinity Labels; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D011485:Protein Binding; D013095:Spermidine / Q000378:metabolism*; D013329:Structure-Activity Relationship
D018528:ATP-Binding Cassette Transporters; D000345:Affinity Labels; D001386:Azides; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C096374:PotA protein, E coli; C096375:PotB protein, E coli; C096376:PotC protein, E coli; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D003545:Cysteine; D013095:Spermidine