Cloning, Expression in Escherichia Coli, and Characterization of Arabidopsis Thaliana UMP/CMP Kinase

Overview

Journal Plant Physiol

Specialty Physiology

Date 1998 May 22

PMID 9576794

Citations 12

Authors

L Zhou

F Lacroute

R Thornburg

Affiliations

Soon will be listed here.

Abstract

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.

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References

Seagrave J, Reyes P . Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: a kinetic analysis of the reaction mechanism. Arch Biochem Biophys. 1987; 254(2):518-25. DOI: 10.1016/0003-9861(87)90132-9. View

Jong A, Kuo C, Campbell J . The CDC8 gene of yeast encodes thymidylate kinase. J Biol Chem. 1984; 259(17):11052-9. View

GALLANGO M, Muller A, Suinaga R . Biochemical characterization of a red cell UMP kinase variant found in the Warao indians of Venezuela. Biochem Genet. 1978; 16(11-12):1085-92. DOI: 10.1007/BF00484529. View

Baranowska H, Zuk J . Chemical mutagenesis and DNA synthesis in cdc8, a DNA replication mutant of Saccharomyces cerevisiae. Curr Genet. 1991; 20(6):471-4. DOI: 10.1007/BF00334774. View

Moller W, Amons R . Phosphate-binding sequences in nucleotide-binding proteins. FEBS Lett. 1985; 186(1):1-7. DOI: 10.1016/0014-5793(85)81326-0. View

Valentin-Hansen P . Uridine-cytidine kinase from Escherichia coli. Methods Enzymol. 1978; 51:308-14. DOI: 10.1016/s0076-6879(78)51041-0. View

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Serina L, Blondin C, Krin E, Sismeiro O, Danchin A, Sakamoto H . Escherichia coli UMP-kinase, a member of the aspartokinase family, is a hexamer regulated by guanine nucleotides and UTP. Biochemistry. 1995; 34(15):5066-74. DOI: 10.1021/bi00015a018. View

Kuo C, Campbell J . Cloning of Saccharomyces cerevisiae DNA replication genes: isolation of the CDC8 gene and two genes that compensate for the cdc8-1 mutation. Mol Cell Biol. 1983; 3(10):1730-7. PMC: 370034. DOI: 10.1128/mcb.3.10.1730-1737.1983. View

10.

Muller-Dieckmann H, Schulz G . Substrate specificity and assembly of the catalytic center derived from two structures of ligated uridylate kinase. J Mol Biol. 1995; 246(4):522-30. DOI: 10.1006/jmbi.1994.0104. View

11.

Gietz D, St Jean A, Woods R, Schiestl R . Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 1992; 20(6):1425. PMC: 312198. DOI: 10.1093/nar/20.6.1425. View

12.

Schricker R, Magdolen V, Kaniak A, Wolf K, Bandlow W . The adenylate kinase family in yeast: identification of URA6 as a multicopy suppressor of deficiency in major AMP kinase. Gene. 1992; 122(1):111-8. DOI: 10.1016/0378-1119(92)90038-q. View

13.

Liljelund P, Lacroute F . Genetic characterization and isolation of the Saccharomyces cerevisiae gene coding for uridine monophosphokinase. Mol Gen Genet. 1986; 205(1):74-81. DOI: 10.1007/BF02428034. View

14.

Newlon C, FANGMAN W . Mitochondrial DNA synthesis in cell cycle mutants of Saccharomyces cerevisiae. Cell. 1975; 5(4):423-8. DOI: 10.1016/0092-8674(75)90061-6. View

15.

Serina L, Bucurenci N, Gilles A, Surewicz W, Fabian H, Mantsch H . Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP. Biochemistry. 1996; 35(22):7003-11. DOI: 10.1021/bi960062v. View

16.

Kawai M, Uchimiya H . Biochemical properties of rice adenylate kinase and subcellular location in plant cells. Plant Mol Biol. 1995; 27(5):943-51. DOI: 10.1007/BF00037022. View

17.

Pai E, Sachsenheimer W, Schirmer R, Schulz G . Substrate positions and induced-fit in crystalline adenylate kinase. J Mol Biol. 1977; 114(1):37-45. DOI: 10.1016/0022-2836(77)90281-9. View

18.

Kozak M . Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell. 1986; 44(2):283-92. DOI: 10.1016/0092-8674(86)90762-2. View

19.

Diederichs K, Schulz G . The refined structure of the complex between adenylate kinase from beef heart mitochondrial matrix and its substrate AMP at 1.85 A resolution. J Mol Biol. 1991; 217(3):541-9. DOI: 10.1016/0022-2836(91)90756-v. View

20.

Wiesmuller L, Noegel A, Barzu O, Gerisch G, Schleicher M . cDNA-derived sequence of UMP-CMP kinase from Dictyostelium discoideum and expression of the enzyme in Escherichia coli. J Biol Chem. 1990; 265(11):6339-45. View