Cloning, expression in Escherichia coli, and characterization of Arabidopsis thaliana UMP/CMP kinase.
A cDNA encoding the Arabidopsis thaliana uridine 5'-monophosphate (UMP)/cytidine 5'-monophosphate (CMP) kinase was isolated by complementation of a Saccharomyces cerevisiae ura6 mutant. The deduced amino acid sequence of the plant UMP/CMP kinase has 50% identity with other eukaryotic UMP/CMP kinase proteins. The cDNA was subcloned into pGEX-4T-3 and expressed as a glutathione S-transferase fusion protein in Escherichia coli. Following proteolytic digestion, the plant UMP/CMP kinase was purified and analyzed for its structural and kinetic properties. The mass, N-terminal sequence, and total amino acid composition agreed with the sequence and composition predicted from the cDNA sequence. Kinetic analysis revealed that the UMP/CMP kinase preferentially uses ATP (Michaelis constant [Km] = 29 microM when UMP is the other substrate and Km = 292 microM when CMP is the other substrate) as a phosphate donor. However, both UMP (Km = 153 microM) and CMP (Km = 266 microM) were equally acceptable as the phosphate acceptor. The optimal pH for the enzyme is 6.5. P1, P5-di(adenosine-5') pentaphosphate was found to be a competitive inhibitor of both ATP and UMP.
Zhou
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Department of Biochemistry and Biophysics, Iowa State University, Ames,Iowa 50011, USA.
Lacroute
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Thornburg
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D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D015226:Dinucleoside Phosphates / Q000494:pharmacology; D004795:Enzyme Stability; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009703:Nucleoside-Phosphate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D010940:Plant Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013552:Swine; D013696:Temperature
D015226:Dinucleoside Phosphates; D010940:Plant Proteins; D012333:RNA, Messenger; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase