Nucleoside diphosphate kinase from Myxococcus xanthus. II. Biochemical characterization.
The gene that encodes the 16-kDa GTP-binding protein from Myxococcus xanthus has been cloned, and its DNA sequence has been determined. The gene has been expressed in Escherichia coli by using the lacZ promoter, and its gene product was overproduced (Muñoz-Dorado, J., Inouye, M., and Inouye, S. (1990) J. Biol. Chem. 265, 2702-2706). The gene product thus overproduced in E. coli was purified to homogeneity by a simple four-step procedure and crystallized. Gel filtration of the purified protein revealed that the protein forms a complex of an apparent molecular weight of 50,000, indicating that it exists as a trimer in the cell. It was found that the purified protein can bind not only GTP, but also equally well the other nucleoside diphosphates and triphosphates with no specificity for either the base or the sugar. Nucleoside monophosphates, Pi, and pyrophosphate do not bind to the protein. In the presence of Mg2+, the protein hydrolyzes nucleoside triphosphates to diphosphates and Pi. However, in the presence of EDTA, most of the phosphate remains bound to the protein. The phosphorylated protein can then transfer the phosphate group to a nucleoside diphosphate to form the corresponding nucleoside triphosphate in the presence of Mg2+. The reaction is reversible, and it is considered to occur by a two-step ping-pong mechanism. These results unambiguously demonstrate that the M. xanthus 16-kDa GTP-binding protein is a nucleoside diphosphate kinase.
Muñoz-Dorado
J
J
Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635.
Inouye
S
S
Inouye
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003460:Crystallization; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000096:biosynthesis; D007202:Indicators and Reagents; D007700:Kinetics; D008970:Molecular Weight; D009231:Myxococcales / Q000201:enzymology* / Q000235:genetics; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010770:Phosphotransferases / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D007202:Indicators and Reagents; D010761:Phosphorus Radioisotopes; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase; D019204:GTP-Binding Proteins