Glycerol-3-phosphate Cytidylyltransferase Research Articles

Characterization of cytidylyltransferase enzyme activity through high performance liquid chromatography

The cytidylyltransferases are a family of enzymes that utilize cytidine 5′-triphosphate (CTP) to synthesize molecules that are typically precursors to membrane phospholipids. The most extensively studied cytidylyltransferase is CTP:phosphocholine cytidylyltransferase (CCT), which catalyzes conversion of phosphocholine and CTP to cytidine diphosphocholine (CDP-choline), a step critical for synthesis of the membrane phospholipid phosphatidylcholine (PC). The current method used to determine catalytic activity of CCT measures production of radiolabeled CDP-choline from 14C-labeled phosphocholine. The goal of this research was to develop a CCT enzyme assay that employed separation of non-radioactive CDP-choline from CTP. A C18 reverse phase column with a mobile phase of 0.1 M ammonium bicarbonate (98%) and acetonitrile (2%) (pH 7.4) resulted in separation of solutions of the substrate CTP from the product CDP-choline. A previously characterized truncated version of rat CCTα (denoted CCTα236) was used to test the HPLC enzyme assay by measuring CDP-choline product formation. The Vmax for CCTα236 was 3850 nmol/min/mg and K0.5 values for CTP and phosphocholine were 4.07 mM and 2.49 mM, respectively. The HPLC method was applied to glycerol 3-phosphate cytidylyltransferase (GCT) and CTP:2-C-methyl-D-erythritol-4-phosphate cytidylyltransferase synthetase (CMS), members of the cytidylyltransferase family that produce CDP-glycerol and CDP-methylerythritol, respectively.

Comparative kinetic analysis of glycerol 3-phosphate cytidylyltransferase from Enterococcus faecalis and Listeria monocytogenes

SummaryBackgroundGlycerol 3-phosphate cytidylyltransferase (GCT) is an enzyme central to the synthesis of teichoic acids, components of the cell wall in gram positive bacteria. Catalysis by GCT from Enterococcus faecalis and Listeria monocytogenes has been investigated and catalytic properties compared.Material/MethodsThe genes encoding GCT were cloned from genomic DNA and recombinant proteins expressed in E. coli and purified. Enzyme assays were used to determine kinetic constants kcat and Km. Chemical crosslinking provided a means to assess quaternary structure of each GCT.ResultsRecombinant Enterococcus faecalis GCT had an apparent kcat value of 1.51 s−1 and apparent Km values of 2.42 mM and 4.03 mM with respect to substrates cytidine 5′-triphosphate (CTP) and glycerol phosphate. Listeria monocytogenes GCT had an apparent kcat value of 4.15 s−1 and apparent Km values of 1.52 mM and 6.56 mM with respect to CTP and glycerol phosphate. This resulted in kcat/Km values of 0.62 s−1mM−1 and 0.37 s−1mM−1 for E. faecalis GCT and 2.73 s−1mM−1 and 0.63 s−1mM−1 for L. monocytogenes GCT with respect to CTP and glycerol phosphate, respectively.ConclusionsThe genome of both Enterococcus faecalis and Listeria monocytogenes contain a gene that encodes a functional GCT. The genes are 67% identical at the nucleotide level and the encoded proteins exhibit a 63% amino acid identity. The purified, recombinant enzymes each appear to be dimeric and display similar kinetic characteristics. Studying the catalytic characteristics of GCT isoforms from pathogenic bacteria provides information important for the future development of potential antibacterial agents.

Archaeal RibL: A New FAD Synthetase That Is Air Sensitive

FAD synthetases catalyze the transfer of the AMP portion of ATP to FMN to produce FAD and pyrophosphate (PP(i)). Monofunctional FAD synthetases exist in eukaryotes, while bacteria have bifunctional enzymes that catalyze both the phosphorylation of riboflavin and adenylation of FMN to produce FAD. Analyses of archaeal genomes did not reveal the presence of genes encoding either group, yet the archaea contain FAD. Our recent identification of a CTP-dependent archaeal riboflavin kinase strongly indicated the presence of a monofunctional FAD synthetase. Here we report the identification and characterization of an archaeal FAD synthetase. Methanocaldococcus jannaschii gene MJ1179 encodes a protein that is classified in the nucleotidyl transferase protein family and was previously annotated as glycerol-3-phosphate cytidylyltransferase (GCT). The MJ1179 gene was cloned and its protein product heterologously expressed in Escherichia coli. The resulting enzyme catalyzes the adenylation of FMN with ATP to produce FAD and PP(i). The MJ1179-derived protein has been designated RibL to indicate that it follows the riboflavin kinase (RibK) step in the archaeal FAD biosynthetic pathway. Aerobically isolated RibL is active only under reducing conditions. RibL was found to require divalent metals for activity, the best activity being observed with Co(2+), where the activity was 4 times greater than that with Mg(2+). Alkylation of the two conserved cysteines in the C-terminus of the protein resulted in complete inactivation. RibL was also found to catalyze cytidylation of FMN with CTP, making the modified FAD, flavin cytidine dinucleotide (FCD). Unlike other FAD synthetases, RibL does not catalyze the reverse reaction to produce FMN and ATP from FAD and PP(i). Also in contrast to other FAD synthetases, PP(i) inhibits the activity of RibL.

Glycerol-3-phosphate Cytidylyltransferase: STRUCTURAL CHANGES INDUCED BY BINDING OF CDP-GLYCEROL AND THE ROLE OF LYSINE RESIDUES IN CATALYSIS

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.

CTP:glycerol 3-phosphate cytidylyltransferase (TarD) from Staphylococcus aureus catalyzes the cytidylyl transfer via an ordered Bi–Bi reaction mechanism with micromolar Km 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 μM, 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 °C) was shown to be xylose-dependent.

Precise deletion of tagD and controlled depletion of its product, glycerol 3-phosphate cytidylyltransferase, leads to irregular morphology and lysis of Bacillus subtilis grown at physiological temperature.

Using a previously reported conditional expression system for use in Bacillus subtilis (A. P. Bhavsar, X. Zhao, and E. D. Brown, Appl. Environ. Microbiol. 67:403-410, 2001), we report the first precise deletion of a teichoic acid biosynthesis (tag) gene, tagD, in B. subtilis. This teichoic acid mutant showed a lethal phenotype when characterized at a physiological temperature and in a defined genetic background. This tagD mutant was subject to full phenotypic rescue upon expression of the complementing copy of tagD. Depletion of the tagD gene product (glycerol 3-phosphate cytidylyltransferase) via modulated expression of tagD from the amyE locus revealed structural defects centered on shape, septation, and division. Thickening of the wall and ultimately lysis followed these events.

Delineation of the allosteric mechanism of a cytidylyltransferase exhibiting negative cooperativity.

The dimeric enzyme CTP:glycerol-3-phosphate cytidylyltransferase (GCT) displays strong negative cooperativity between the first and second binding of its substrate, CTP. Using NMR to study the allosteric mechanism of this enzyme, we observe widespread chemical shift changes for the individual CTP binding steps. Mapping these changes onto the molecular structure allowed the formulation of a detailed model of allosteric conformational change. Upon the second step of ligand binding, NMR experiments indicate an extensive loss of conformational exchange broadening of the backbone resonances of GCT. This suggests that a fraction of the free energy of negative cooperativity is entropic in origin.

Negative Cooperativity of Substrate Binding but Not Enzyme Activity in Wild-type and Mutant Forms of CTP:Glycerol-3-Phosphate Cytidylyltransferase

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.

Crystallization and preliminary X-ray diffraction studies of glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus.

Glycerol 3-phosphate cytidylyltransferase from Staphylococcus aureus (TarD(Sa)) has been expressed in Escherichia coli, purified to homogeneity and crystallized. The strategy used for determining crystallization conditions employed hanging-drop sparse-matrix screens and required a combination of three different optimization approaches. Specifically, the presence or absence of cofactors needed to be surveyed, the effects of small-molecule additives required exploration and the rate of vapour-diffusion had to be varied in order to obtain crystals of TarD(Sa) suitable for diffraction studies. Crystals thus obtained belong to the space group P3(1)21, with unit-cell parameters a = b = 92.2, c = 156.1 A, and contain four TarD(Sa) molecules per asymmetric unit. The resolution limit observed for these crystals using synchrotron radiation is 3.0 A.

Identification of Functional Conserved Residues of CTP:glycerol-3-phosphate Cytidylyltransferase: ROLE OF HISTIDINES IN THE CONSERVED HXGH IN CATALYSIS

The CTP:glycerol-3-phosphate cytidylyltransferase (GCT) of Bacillus subtilis has been shown to be similar in primary structure to the CTP:phosphocholine cytidylyltransferases of several organisms. To identify the residues of this cytidylyltransferase family that function in catalysis, the conserved hydrophilic amino acid residues plus a conserved tryptophan of the GCT were mutated to alanine. The most dramatic losses in activity occurred with H14A and H17A; these histidine residues are part of an HXGH sequence similar to that found in class I aminoacyl-tRNA synthetases. The kcat values for H14A and H17A were decreased by factors of 5 x 10(-5) and 4 x 10(-4), respectively, with no significant change in Km values. Asp-11, which is found near the HXGH sequence in the cytidylyltransferases but not aminoacyl-tRNA synthetases, was also important for activity, with the D11A mutation decreasing activity by a factor of 2 x 10(-3). Several residues found in the sequence RTEGISTT, a signature sequence for this cytidylyltransferase family, as well as other isolated residues were also shown to be important for activity, with kcat values decreasing by factors of 0.14-4 x 10(-4). The Km values of three mutant enzymes, D38A, W74A, and D94A, for both CTP and glycerol-3-phosphate were 6-130-fold higher than that of the wild-type enzyme. Mutant enzymes were analyzed by two-dimensional NMR to determine if the overall structures of the enzymes were intact. One of the mutant enzymes, D66A, was defective in overall structure, but several of the others, including H14A and H17A, were not. These results indicate that His-14 and His-17 play a role in catalysis and suggest that their role is similar to the role of the His residues in the HXGH sequence in class I aminoacyl-tRNA synthetases, i.e. to stabilize a pentacoordinate transition state.