Expression, purification, and characterization of CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis.
Bacillus subtilis contains the gene for CTP:glycerol-3-phosphate cytidylyltransferase, which is involved in biosynthesis of the major teichoic acid of the B. subtilis cell wall. When this gene was expressed in Escherichia coli under the control of the T7 promoter, the glycerol-3-phosphate cytidylyltransferase accumulated to a level of about 15% of cellular protein. The expressed glycerol-3-phosphate cytidylyltransferase was purified to homogeneity by ion-exchange chromatography, gel filtration, and affinity chromatography on blue Sepharose. Approximately 47 mg of pure enzyme was obtained from a 660-ml culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the subunit molecular weight of the purified enzyme was about 15,000. The molecular weight of the native enzyme was found to be 30,900 by gel filtration analysis, suggesting that the native enzyme is a homodimer. The pH optimum was very broad, from 6.5 to 9.5, and the enzyme was stable at alkaline conditions. A divalent cation, either Co2+, Mg2+, Mn2+, or Fe2+, was required for enzyme activity. Km values for CTP and glycerol 3-phosphate were 3.85 and 3.23 mM, respectively, and the Vmax was 185 units/mg of protein. Initial rate studies and product inhibition patterns indicated that the enzyme catalyzes the reaction by means of a rapid eqilibrium random order mechanism. The availability of large amounts of glycerol-3-phosphate cytidylyltransferase will facilitate enzymological and structural studies on this model cytidylyltransferase.
Park
Y S
YS
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Sweitzer
T D
TD
Dixon
J E
JE
Kent
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001412:Bacillus subtilis / Q000201:enzymology*; D019916:Choline-Phosphate Cytidylyltransferase; D003001:Cloning, Molecular; D003570:Cytidine Triphosphate / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D008670:Metals; D011994:Recombinant Proteins; D003570:Cytidine Triphosphate; D009713:Nucleotidyltransferases; D019916:Choline-Phosphate Cytidylyltransferase; C020675:glycerol-3-phosphate cytidylyltransferase