Properties of the periplasmic ModA molybdate-binding protein of Escherichia coli.
The modABCD operon, located at 17 min on the Escherichia coli chromosome, encodes the protein components of a high affinity molybdate uptake system. Sequence analysis of the modA gene (GenBank L34009) predicts that it encodes a periplasmic binding protein based on the presence of a leader-like sequence at its N terminus. To examine the properties of the ModA protein, the modA structural gene was overexpressed, and its product was purified. The ModA protein was localized to the periplasmic space of the cell, and it was released following a gentle osmotic shock. The N-terminal sequence of ModA confirmed that a leader region of 24 amino acids was removed upon export from the cell. The apparent size of ModA is 31.6 kDa as determined by gel sieve chromatography, whereas it is 22.5 kDa when examined by SDS-polyacrylamide gel electrophoresis. A ligand-dependent protein mobility shift assay was devised using a native polyacrylamide gel electrophoresis protocol to examine binding of molybdate and other anions to the ModA periplasmic protein. Whereas molybdate and tungstate were bound with high affinity (approximately 5 microM), sulfate, chromate, selenate, phosphate, and chlorate did not bind even when tested at 2 mM. A UV spectral assay revealed apparent Kd values of binding for molybdate and tungstate of 3 and 7 microM, respectively. Strains defective in the modA gene were unable to transport molybdate unless high levels of the anion were supplied in the medium. Therefore the modA gene product is essential for high affinity molybdate uptake by the cell. Tungstate interference of molybdate acquisition by the cell is apparently due in part to the high affinity of the ModA protein for this anion.
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Department of Microbiology and Molecular Genetics, University of California, Los Angeles 90095, USA.
Wolin
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Gunsalus
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D000595:Amino Acid Sequence; D000838:Anions; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006868:Hydrolysis; D008969:Molecular Sequence Data; D033902:Periplasmic Binding Proteins; D010641:Phenotype; D011485:Protein Binding; D011499:Protein Processing, Post-Translational
D000838:Anions; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C098002:ModA protein, E coli; D033902:Periplasmic Binding Proteins