Functional cloning of a nucleoside diphosphate kinase from Dictyostelium discoideum.
A lambda gt11 cDNA library from Dictyostelium discoideum was screened by direct labeling of filter replicas with [35S]guanosine 5'-O-(thiotriphosphate) (GTP gamma S). A positive clone was obtained and used as probe to isolate additional clones from which a complete cDNA sequence was determined. The cDNA hybridizes to a single copy gene that is expressed as a 0.6-kilobase mRNA in vegetatively growing amoeba. The open reading frame encodes a protein of 155 amino acids (calculated Mr 16,775), devoid of cysteine residues. The protein contains most of the short consensus motifs characteristic of the catalytic domain of protein kinases although the overall homology with this class of enzymes is not greater than 20%. Its size and amino acid composition indicated that it could be the monomer of a nucleoside diphosphate (NDP) kinase, an enzyme which catalyzes the phosphate transfer from triphospho- to diphosphonucleotides. Indeed, specific NDP kinase activity was found in extracts of bacteria transformed with a plasmid expressing the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the extracts incubated in the presence of [35S]GTP gamma S revealed a single 35S-labeled band of size corresponding to the protein, which likely represents the stable thiophosphorylated reaction intermediate characteristic for the ping-pong reaction mechanism of NDP kinases. The formation of this labeled intermediate probably allowed the detection of the enzyme on the filters during the screening procedure. Although NDP kinases from a great variety of sources have been characterized, the primary structure of the D. discoideum NDP kinase is the first reported for an enzyme of eukaryotic origin.
Lacombe
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Unité de Biochimie Cellulaire, Institut Pasteur, Paris, France.
Wallet
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Troll
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Véron
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics* / Q000378:metabolism; D015345:Oligonucleotide Probes; D010770:Phosphotransferases / Q000235:genetics*; D010957:Plasmids; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D015345:Oligonucleotide Probes; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D010770:Phosphotransferases; D009701:Nucleoside-Diphosphate Kinase