Structure and Mechanism of CTP:Phosphocholine Cytidylyltransferase (LicC) from Streptococcus pneumoniae

Abstract

Pneumococcal LicC is a member of the nucleoside triphosphate transferase superfamily and catalyzes the transfer of a cytidine monophosphate from CTP to phosphocholine to form CDP-choline. The structures of apoLicC and the LicC⅐CDP-choline⅐Mg 2؉ ternary complex were determined, and the comparison of these structures reveals a significant conformational change driven by the multivalent coordination of Mg 2؉ . The key event is breaking the Glu 216 ⅐Arg 129 salt bridge, which triggers the coalescence of four individual ␤-strands into two extended ␤-sheets. These movements reorient the side chains of Trp 136 and Tyr 190 for the optimal binding and alignment of the phosphocholine moiety. Consistent with these conformational changes, LicC operates via a compulsory ordered kinetic mechanism. The structures explain the substrate specificity of LicC for CTP and phosphocholine and implicate a direct role for Mg 2؉ in aligning phosphocholine for in-line nucleophilic attack and stabilizing the negative charge that develops in the pentacoordinate transition state. These results provide a structural basis for assigning a specific role for magnesium in the catalytic mechanism of pneumococcal LicC.Choline metabolism plays a key role in cell separation, transformation, autolysis, and pathogenicity of Streptococcus pneumoniae. The only known metabolic fate of choline is to decorate the teichoic and lipoteichoic acids of the cell wall, and choline is an essential nutrient for S. pneumoniae (1). The cell surface P-Cho 1 facilitates the interaction with the host surface and induces attachment and invasion (2, 3). The importance of choline in pathogenesis is not confined to S. pneumoniae but also plays a role in Hemophilus influenzae (4 -7), Pseudomonas aeruginosa, and Neisseria gonorrhoeae (8,9). In addition, cell wall P-Cho serves as the scaffold for a group of choline-binding proteins that are secreted from the cells and subsequently attached to the cell surface by their homologous choline-binding domains (see Ref. 10 and references therein). These cholinebinding proteins are essential for many aspects of S. pneumoniae cell physiology including competence and stationary phase lysis.The pathway for choline metabolism in S. pneumoniae and H. influenzae has been hypothesized to consist of a choline transport system, a choline kinase, CTP:phosphocholine cytidylyltransferase (CCT), and a choline phosphotransferase that transfers P-Cho from CDP-Cho to either lipoteichoic acid or lipopolysaccharide (5). The existence of this pathway is supported by the detection of choline kinase and CCT activity in crude extracts of S. pneumoniae (11,12). Genetic elements required for choline incorporation into the lipopolysaccharide of H. influenzae are found in the lic1 locus, which contains four open reading frames. The hypothesis drawn from the bioinformatic analysis of the lic1 locus (4) is that licA corresponds to choline kinase based on a 31% identity to the choline kinase of Saccharomyces cerevisiae over the short span of 40 amino acids between r...

The Kennedy pathway—De novo synthesis of phosphatidylethanolamine and phosphatidylcholine

“…The Streptococcus pneumoniae LicC gene, despite showing no sequence homology to eukaryotic CCTs, does form CDPcholine, which is required for cell-wall elaboration of cholinephosphate (73). The LicC gene and the corresponding LicA gene, which encodes CK activity, have been noted in several other pathogenic Gram-negative bacteria, that is, Treponema denticola (74).…”

Crystal Structure of Archaeoglobus fulgidus CTP:Inositol-1-Phosphate Cytidylyltransferase, a Key Enzyme for Di- myo -Inositol-Phosphate Synthesis in (Hyper)Thermophiles

“…The enzyme shares structural homology with other pyrophosphorylases showing the canonical motif G-X-G-T-(R/S)-X 4 -P-K. CTP, L-myo-inositol-1-phosphate, and CDP-inositol were docked into the catalytic site, which provided insights into the binding mode and high specificity of the enzyme for CTP. This work is an important step toward the final goal of understanding the full catalytic route for DIP synthesis in the native, bifunctional enzyme.Enzymes classified in the nucleoside triphosphate-transferase family (PF00483) typically transfer nucleoside monophosphate (NMP) from nucleoside triphosphates (NTP) to an acceptor phosphoryl group belonging to a small molecule such as phosphocholine, hexose-1-phosphate, or ribitol-5-phosphate (2,16,20). This activity leads to release of pyrophosphate and production of a nucleoside diphospho-acceptor that is subsequently utilized by glycosyltransferases in a myriad of reactions of vital importance for the cellular functions.…”

“…Enzymes classified in the nucleoside triphosphate-transferase family (PF00483) typically transfer nucleoside monophosphate (NMP) from nucleoside triphosphates (NTP) to an acceptor phosphoryl group belonging to a small molecule such as phosphocholine, hexose-1-phosphate, or ribitol-5-phosphate (2,16,20). This activity leads to release of pyrophosphate and production of a nucleoside diphospho-acceptor that is subsequently utilized by glycosyltransferases in a myriad of reactions of vital importance for the cellular functions.…”

Different Pathways of Choline Metabolism in Two Choline-Independent Strains ofStreptococcus pneumoniaeand Their Impact on Virulence

“…The protein products of licB, licA, and licC catalyze cellular uptake (5) and conversion of intracellular choline to phosphorylcholine in an ATP-dependent reaction (28) followed by the conversion of phosphorylcholine in a CTP-dependent reaction to CDP-choline (2,14,17), which is assumed to be the substrate of an additional enzyme(s) that catalyzes the incorporation of P-choline residues (one or two, depending on the particular S. pneumoniae strain) into the teichoic acid chains. The genetic determinants involved appear to be part of a second operon (lic2) adjacent to lic1 on the pneumococcal chromosome (11,12,31).…”