Cloning, expression in Escherichia coli, and characterization of Arabidopsis thaliana UMP/CMP kinase.
Ключови думи
Резюме
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.