The x-ray structure analysis of CTP:Glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis.

Abstract

Cytidylyltransferases are enzymes that activate intermediates in a wide range of biochemical reactions. In mammals, they play a key role in phospholipid and glycoprotein biosynthesis. In particular, phosphocholine cytidylyltransferase (CCTase) catalyzes the rate limiting step in biosynthesis of phosphatidylcholine, the major membrane phospholipid of eukaryotes. A bacterial cytidylyltransferase, glycerol-3-phosphate cytidylyltransferase (GCTase) from B. subtilis, has proven to be a useful model for the study of more complex mammalian cytidylyltransferases. The level of homology between GCTase and the catalytic region of the CCTases predicts that the three dimensional structures will be similar. The structure of GCTase has been determined by the method of multiwavelength anomalous diffraction, and partly refined with data from each of two crystal forms. Monoclinic crystals, space group P2$\sb1$ (a = 44.5A, b = 61.4A, c = 56.4A, $\beta=113.1\sp\circ),$ and rhombohedral crystals, R3 (a = b = 143.5A, c = 41.7A), were grown using polyethylene glycol as a precipitant. The monoclinic crystals display variable degrees of merohedral twinning. Multiwavelength diffraction data from a mercury derivatized R3 crystal were used for initial phasing; density modification and noncrystallographic symmetry averaging improved the quality of the electron density map. Refinement of the partial model (125 of 129 residues) versus 2.0 A resolution data from the monoclinic crystal form has led to an R-factor of 21.4% and a free R-factor of 30.5%. GCTase is a homodimer, each monomer consisting of a single $\alpha/\beta$ domain which is similar to the classical Rossmann nucleotide binding fold. The x-ray analysis of GCTase has revealed cytidine triphosphate (CTP), one of the substrates, bound at each of the active sites of the homodimer. Two conserved sequences, the HxGH$\sp{17}$ and RTEGISTT$\sp{120}$ motifs, form part of the binding site for CTP. The presence of the HxGH motif in GCTase had suggested similarities with the class I amino acyl-tRNA synthetases. The structure of GCTase indeed resembles the nucleotide binding domain of the amino acyl-tRNA synthetases, and furthermore, the nucleotides are bound in a similar fashion in both structures.