A prototypical cytidylyltransferase: CTP : glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis

Background: The formation of critical intermediates in the biosynthesis of lipids and complex carbohydrates is carried out by cytidylyltransferases, w hich utilize CTP to form activated CDP-alcohols or CMP-acid sugars plus ino rganic pyrophosphate. Several cytidylyltransferases are related and constit ute a conserved family of enzymes. The eukaryotic members of the family are complex enzymes with multiple regulatory regions or repeated catalytic dom ains, whereas the bacterial enzyme, CTP:glycerol-3-phosphate cytidylyltrans ferase (GGT), contains only the catalytic domain. Thus, GCT provides an exc ellent model for the study of catalysis by the eukaryotic cytidylyltransfer ases. Results: The crystal structure of GCT from Bacillus subtilis has been deter mined by multiwavelength anomalous diffraction using a mercury derivative a nd refined to 2.0 Angstrom resolution (R-factor 0.196; R-factor 0.255). GCT is a homodimer; each monomer comprises an alpha/beta fold with a central 3 -2-1-4-5 parallel beta sheet. Additional helices and loops extending from t he alpha/beta core form a bowl that binds substrates. CTP, bound at each ac tive site of the homodimer, interacts with the conserved (14)HXGH and (113) RTXGISTT motifs. The dimer interface incorporates part of a third motif, (R YVDEVI)-R-63, and includes hydrophobic residues adjoining the HXGH sequence . Conclusions: Structure superpositions relate GCT to the catalytic domains f rom class I aminoacyl-tRNA synthetases, and thus expand the tRNA synthetase family of folds to include the catalytic domains of the family of cytidyly ltransferases. GCT and aminoacyl-tRNA synthetases catalyze analogous reacti ons, bind nucleotides in similar U-shaped conformations, and depend on hist idines from analogous HXGH motifs for activity. The structural and other si milarities support proposals that GCT, like the synthetases, catalyzes nucl eotidyl transfer by stabilizing a pentavatent transition state at the alpha -phosphate of CTP.