PMID:7867647
| Citation |
de Pina, K, Navarro, C, McWalter, L, Boxer, DH, Price, NC, Kelly, SM, Mandrand-Berthelot, MA and Wu, LF (1995) Purification and characterization of the periplasmic nickel-binding protein NikA of Escherichia coli K12. Eur. J. Biochem. 227:857-65 |
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| Abstract |
The nik operon of Escherichia coli encodes a periplasmic binding-protein-dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel-binding protein NikA by cloning the nikA gene into an overexpression vector, pRE1. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion-exchange chromatography. N-terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel-binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence. NikA binds one atom of nickel/molecule of protein with a dissociation constant (Kd) of less than 0.1 microM. Other metals (cobalt, copper, iron) are bound at least 10-fold less tightly. The high specificity for Ni2+ is also demonstrated by high-performance immobilized-metal-ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 microM Ni2+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild-type strain MC4100 contains about 23,000 molecules of NikA/cell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar-dependent repellent chemotaxis. |
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| Keywords |
Bacterial Proteins; Base Sequence; Biological Transport, Active; Chemotaxis; Cloning, Molecular; DNA Topoisomerases, Type I/genetics; DNA Topoisomerases, Type I/isolation & purification; DNA Topoisomerases, Type I/metabolism; DNA, Bacterial/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Escherichia coli/metabolism; Genes, Bacterial; Genetic Vectors; Metals/metabolism; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Nickel/metabolism; Operon |
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