cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli.
A cDNA coding for UMP-CMP kinase from Dictyostelium discoideum was isolated from a lambda gt11 expression library and sequenced. The corresponding mRNA has a size of 0.7 kilobase and is down-regulated during early development of D. discoideum. Southern blotting demonstrated that the UMP-CMP kinase is encoded by a single gene. The deduced amino acid sequence of UMP-CMP kinase shows a high degree of homology with adenylate kinases from different sources with the highest degree of homology to cytosolic adenylate kinase from vertebrate muscle (43%). The enzyme expressed in Escherichia coli after cloning the cDNA into an ATG expression vector was purified and analyzed for its structural and kinetic properties. The UMP-CMP kinase uses preferentially ATP (Km,app = 25 microM) as phosphate donor and is specific for UMP (Km,app = 0.4 mM) and CMP (Km,app = 0.1 mM). The enzyme is strongly inhibited by the substrate analogue P1-(adenosine-5')-P5-(uridine-5')-pentaphosphate (Ki between 0.05 and 0.1 microM) and is inactivated by modification of free thiol groups with 5,5'-dithiobis(2-nitrobenzoic acid).
Wiesmüller
L
L
Max-Planck-Institut für Biochemie, Martinsried, Federal Republic of Germany.
Noegel
A A
AA
Bârzu
O
O
Gerisch
G
G
Schleicher
M
M
D000263:Adenylate Kinase / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics* / Q000302:isolation & purification; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009703:Nucleoside-Phosphate Kinase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010770:Phosphotransferases / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D011994:Recombinant Proteins; D010770:Phosphotransferases; C019979:cytidylate kinase; D000263:Adenylate Kinase; D009703:Nucleoside-Phosphate Kinase