Publications

• Expression, purification, and characterization of CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis.

  Park Y.S., Sweitzer T.D., Dixon J.E., Kent C.

  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 cytidyly ... >> More

  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. << Less

  J. Biol. Chem. 268:16648-16654(1993) [PubMed] [EuropePMC]

• 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.

  Badurina D.S., Zolli-Juran M., Brown E.D.

  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 cytidylyltransfe ... >> More

  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. << Less

  Biochim. Biophys. Acta 1646:196-206(2003) [PubMed] [EuropePMC]

• Negative cooperativity of substrate binding but not enzyme activity in wild-type and mutant forms of CTP:glycerol-3-phosphate cytidylyltransferase.

  Sanker S., Campbell H.A., Kent C.

  CTP:glycerol-3-phosphate cytidylyltransferase (GCT) catalyzes the synthesis of CDP-glycerol for teichoic acid biosynthesis in certain Gram-positive bacteria. This enzyme is a model for a cytidylyltransferase family that includes the enzymes that synthesize CDP-choline and CDP-ethanolamine for phos ... >> More

  CTP:glycerol-3-phosphate cytidylyltransferase (GCT) catalyzes the synthesis of CDP-glycerol for teichoic acid biosynthesis in certain Gram-positive bacteria. This enzyme is a model for a cytidylyltransferase family that includes the enzymes that synthesize CDP-choline and CDP-ethanolamine for phosphatidylcholine and phosphatidylethanolamine biosynthesis. We have used quenching of intrinsic tryptophan fluorescence to measure binding affinities of substrates to the GCT from Bacillus subtilis. Binding of either CTP or glycerol-3-phosphate to GCT was biphasic, with two binding constants of about 0.1-0.3 and 20-40 microm for each substrate. The stoichiometry of binding was 2 molecules of substrate/enzyme dimer, so the two binding constants represented distinctly different affinities of the enzyme for the first and second molecule of each substrate. The biphasic nature of binding was observed with the wild-type GCT as well as with several mutants with altered Km or kcat values. This negative cooperativity of binding was also seen when a catalytically defective mutant was saturated with two molecules of CTP and then titrated with glycerol-3-phosphate. Despite the pronounced negative cooperativity of substrate binding, negative cooperativity of enzyme activity was not observed. These data support a mechanism in which catalysis occurs only when the enzyme is fully loaded with 2 molecules of each substrate/enzyme dimer. << Less

  J. Biol. Chem. 276:37922-37928(2001) [PubMed] [EuropePMC]

• Glycerol-3-phosphate cytidylyltransferase. Structural changes induced by binding of CDP-glycerol and the role of lysine residues in catalysis.

  Pattridge K.A., Weber C.H., Friesen J.A., Sanker S., Kent C., Ludwig M.L.

  The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to fo ... >> More

  The bacterial enzyme, glycerol-3-phosphate cytidylyltransferase (GCT), is a model for mammalian cytidylyltransferases and is a member of a large superfamily of nucleotidyltransferases. Dimeric GCT from Bacillus subtilis displays unusual negative cooperativity in substrate binding and appears to form products only when both active sites are occupied by substrates. Here we describe a complex of GCT with the product, CDP-glycerol, in a crystal structure in which bound sulfate serves as a partial mimic of the second product, pyrophosphate. Binding of sulfate to form a pseudo-ternary complex is observed in three of the four chains constituting the asymmetric unit and is accompanied by a backbone rearrangement at Asp11 and ordering of the C-terminal helix. Comparison with the CTP complex of GCT, determined previously, reveals that in the product complex the active site closes around the glycerol phosphate moiety with a concerted motion of the segment 37-47 that includes helix B. This rearrangement allows lysines 44 and 46 to interact with the glycerol and cytosine phosphates of CDP-glycerol. Binding of CDP-glycerol also induces smaller movements of residues 92-100. Roles of lysines 44 and 46 in catalysis have been confirmed by mutagenesis of these residues to alanine, which decreases Vmax(app) and has profound effects on the Km(app) for glycerol-3-phosphate. << Less

  J. Biol. Chem. 278:51863-51871(2003) [PubMed] [EuropePMC]