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Cloning, Expression in Escherichia coli, and Characterization of Arabidopsis thaliana UMP/CMP Kinase1

Lan Zhou, François Lacroute, and Robert Thornburg*

Department of Biochemistry and Biophysics, Iowa State University, Ames, Iowa 50011 (L.Z., R.T.); and Centre de Génétique Moléculaire, Centre Nationale de La Recherche Scientifique, 22 Avenue de la Terrace, 91198 Gif sur Yvette, cedex France (F.L., R.T.)

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 µm when UMP is the other substrate and Km = 292 µm when CMP is the other substrate) as a phosphate donor. However, both UMP (Km = 153 µm) and CMP (Km = 266 µm) 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.

1   This research was supported by grant no. 91-37301-6208 from the U.S. Department of Agriculture. This is paper no. J-17416 from the Iowa Agriculture and Home Economics Experiment Station.
*   Corresponding author; e-mail thorn{at}iastate.edu; fax 1-515-294-0453.

Plant Physiol. (1998) 117: 245-254
Copyright Clearance Center:   0032-0889/98/117/0245/10
© 1998 American Society of Plant Physiologists




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