Background: The formation of critical intermediates in the biosynthesis of lipids and complex carbohydrates is carried out by cytidylyltransferases, which utilize CTP to form activated CDP-alcohols or CMP-acid sugars plus inorganic pyrophosphate. Several cytidylyltransferases are related and constitute a conserved family of enzymes. The eukaryotic members of the family are complex enzymes with multiple regulatory regions or repeated catalytic domains, whereas the bacterial enzyme, CTP:glycerol-3-phosphate cytidylyltransferase (GCT), contains only the catalytic domain. Thus, GCT provides an excellent model for the study of catalysis by the eukaryotic cytidylyltransferases.
Results: The crystal structure of GCT from Bacillus subtilis has been determined by multiwavelength anomalous diffraction using a mercury derivative and refined to 2.0 A resolution (R(factor) 0.196; R(free) 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 the alpha/beta core form a bowl that binds substrates. CTP, bound at each active site of the homodimer, interacts with the conserved (14)HXGH and (113)RTXGISTT motifs. The dimer interface incorporates part of a third motif, (63)RYVDEVI, and includes hydrophobic residues adjoining the HXGH sequence.
Conclusions: Structure superpositions relate GCT to the catalytic domains from class I aminoacyl-tRNA synthetases, and thus expand the tRNA synthetase family of folds to include the catalytic domains of the family of cytidylyltransferases. GCT and aminoacyl-tRNA synthetases catalyze analogous reactions, bind nucleotides in similar U-shaped conformations, and depend on histidines from analogous HXGH motifs for activity. The structural and other similarities support proposals that GCT, like the synthetases, catalyzes nucleotidyl transfer by stabilizing a pentavalent transition state at the alpha-phosphate of CTP.