CTP:glycerol 3-phosphate cytidylyltransferase (TarD) from Staphylococcus aureus catalyzes the cytidylyl transfer via an ordered Bi-Bi reaction mechanism with micromolar K(m) values.
CTP:glycerol 3-phosphate cytidylyltransferase catalyzes the formation of CDP-glycerol, an activated form of glycerol 3-phosphate and key precursor to wall teichoic acid biogenesis in Gram-positive bacteria. There is high sequence identity (69%) between the CTP:glycerol 3-phosphate cytidylyltransferases from Bacillus subtilis 168 (TagD) and Staphylococcus aureus (TarD). The B. subtilis TagD protein was shown to catalyze cytidylyltransferase via a random mechanism with millimolar K(m) values for both CTP and glycerol 3-phosphate [J. Biol. Chem. 268, (1993) 16648] and exhibited negative cooperativity in the binding of substrates but not in catalysis [J. Biol. Chem. 276, (2001) 37922]. In the work described here on the S. aureus TarD protein, we have elucidated a steady state kinetic mechanism that is markedly different from that determined for B. subtilis TagD. Steady state kinetic experiments with recombinant, purified TarD employed a high-performance liquid chromatography assay developed in this work. The data were consistent with a ternary complex model. The K(m) values for CTP and glycerol 3-phosphate were 36 and 21 microM, respectively, and the k(cat) was 2.6 s(-1). Steady state kinetic analysis of the reverse (pyrophosphorylase) reaction was also consistent with a ternary complex model. Product inhibition studies indicated an ordered Bi-Bi reaction mechanism where glycerol 3-phosphate was the leading substrate and the release of CDP-glycerol preceded that of pyrophosphate. Finally, we investigated the capacity of S. aureus tarD to substitute for tagD in B. subtilis. The tarD gene was placed under control of the xylose promoter in a B. subtilis 168 mutant defective in tagD (temperature-sensitive, tag-12). Growth of the resulting strain at the restrictive temperature (47 degrees C) was shown to be xylose-dependent.
Badurina
David S
DS
Antimicrobial Research Centre, Department of Biochemistry, McMaster University, Room 4H2, 1200 Main St. West, Hamilton, Ontario, Canada L8N 3Z5.
Zolli-Juran
Michela
M
Brown
Eric D
ED
D002851:Chromatography, High Pressure Liquid; D003570:Cytidine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D005994:Glycerophosphates / Q000096:biosynthesis / Q000378:metabolism; D008433:Mathematics; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011755:Pyrophosphatases / Q000737:chemistry / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology*; D013379:Substrate Specificity; D013682:Teichoic Acids / Q000378:metabolism
D005994:Glycerophosphates; D009702:Nucleoside Diphosphate Sugars; D013682:Teichoic Acids; D003570:Cytidine Triphosphate; C100068:cytidine diphosphate glycerol; C029620:alpha-glycerophosphoric acid; D009713:Nucleotidyltransferases; C020675:glycerol-3-phosphate cytidylyltransferase; D011755:Pyrophosphatases