The CTP:phosphocholine cytidylyltransferase encoded by the licC gene of Streptococcus pneumoniae: cloning, expression, purification, and characterization.
Streptococcus pneumoniae is a member of a small group of bacteria that display phosphocholine on the cell surface, covalently attached to the sugar groups of teichoic acid and lipoteichoic acid. The putative pathway for this phosphocholine decoration is, in its first two enzymes, functionally similar to the CDP-choline pathway used for phosphatidylcholine biosynthesis in eukaryotes. We show that the licC gene encodes a functional CTP:phosphocholine cytidylyltransferase (CCT). The enzyme has been expressed and purified to homogeneity. Assay conditions were optimized, particularly with respect to linearity with time, pH, Mg(2+), and ammonium sulfate concentration. The pure enzyme has K(M) values of 890+/-240 microM for CTP, and 390+/-170 microM for phosphocholine. The k(cat) is 17.5+/-4.0 s(-1). S. pneumoniae CTP:phosphocholine cytidylyltransferase (SpCCT) is specific for CTP or dCTP as the nucleotide substrate. SpCCT is strongly inhibited by Ca(2+). The IC(50) values for recombinant and native SpCCT are 0.32+/-0.04 and 0.27+/-0.03 mM respectively. The enzyme is also inhibited by all other tested divalent cations, including Mg(2+) at high concentrations. The cloning and expression of this enzyme sets the stage for design of inhibitors as possible antipneumococcal drugs.
Campbell
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Department of Biological Chemistry, 4417 Medical Science I, University of Michigan Medical Center, Ann Arbor, MI 48109-0606, USA.
Kent
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D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D002118:Calcium / Q000494:pharmacology; D002413:Cations, Divalent; D019916:Choline-Phosphate Cytidylyltransferase / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D008274:Magnesium; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013296:Streptococcus pneumoniae / Q000201:enzymology / Q000235:genetics*
D002413:Cations, Divalent; D019916:Choline-Phosphate Cytidylyltransferase; D008274:Magnesium; D000645:Ammonium Sulfate; D002118:Calcium