X-Ray Crystallographic Studies of Glycerol-3-Phosphate Cytidylyltransferase from Staphylococcus Aureus

Abstract

Glycerol-3-phosphate cytidylyltransferase from 𝘚𝘵𝘢𝘱𝘩𝘺𝘭𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘢𝘶𝘳𝘦𝘶𝘴 complexed with CTP (TarDₛₐ-CTP) was crystallized by the hanging drop-vapor diffusion method at 22°C. Determination of crystallization condition included examination of the amount of precipitant, investigation of the effects of small molecules, and alteration of the rate of diffusion. With these three optimization steps, crystals suitable for x-ray diffraction study were produced. During data processing, TarDₛₐ-CTP was determined to belong to the space group P3₁21, with unit-cell dimensions a=b=92.2 and c=156.1Å. The crystal structure of TarDₛₐ-CTP was solved to 3.0Å by molecular replacement, using TagD from 𝘉𝘢𝘤𝘪𝘭𝘭𝘶𝘴 𝘴𝘶𝘣𝘵𝘪𝘭𝘪𝘴 as a search model. Unlike the search model, TarDₛₐ appears as a tetramer in the asymmetric unit. This result also confirms the gel-filtration and ultracentrifugation studies that were done previously. Although TarDₛₐ crystals were grown in the presence of CTP, the crystal structure does not reveal convincing data for the location and position of this co-factor. However, the data suggests a possible location for CTP in one of the four subunits in an orientation that differs from that of TagD_Bₛ. Unfortunately, the resolution of this data set at 3.0Å is not high enough to corroborate this finding.