W0153

Glycerol-3-Phosphate Cytidylyltransferase: A Novel Target for Drug Design in Staphylococcus aureus. Desiree H. Fong¹, Veronica C.-N. Yim³ & Albert M. Berghuis^1,2, Depts. of ¹Biochemistry and ²Microbiology & Immunology, McGill Univ., Montreal, Quebec, Canada, ³Antimicrobial Research Centre, Dept. of Biochemistry, McMaster Univ., Hamilton, Ontario, Canada.

Glycerol-3-phosphate cytidylyltransferase from Staphylococcus aureus (TarD_Sa) is involved in cell wall biosynthesis and deduced to be essential to bacterial survival. Here we present the apo structure of TarD_Sa.

Due to the extensive use of antibiotics, pathogenic bacteria have evolved the ability to render many of these drugs ineffective, giving rise to Multidrug Resistant Staphylococcus aureus (MRSA). For many gram-positive bacteria, cell wall biosynthesis has been categorized as the primary choice of therapeutic target.

TarD_Sa is 132 amino acid residues long and it is involved in the formation of the linkage unit of the cell wall. This enzyme catalyzes the transfer of the cytosine monophosphate group from CTP to glycerol-3-phosphate, forming CDP-glycerol and pyrophosphate. Rod-shaped crystals of TarD_Sa were grown in the presence of CTP. These crystals belonged to space group P3₁21 with cell dimensions a=b=92.2Å, c=156.1Å. The TarD_Sa structure was solved by molecular replacement using glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis (TagD_Bs) (PDB code:1coz) as the search model. TarD_Sa and TagD_Bs are functionally related proteins sharing 69% sequence identity, although differing in their enzymatic mechanisms. The structure has been refined to 3.0Å with R=0.24 and R_free=0.27.

The structure reveals that four molecules of TarD_Sa exist in each asymmetric unit, forming a dimer of dimers. However, no CTP was observed in the active sites of the enzyme despite the fact that TarD_Sa was incubated with the substrate during crystallization. It has been inferred that CTP binds to conserved regions of the enzyme, including the C-terminus. In the absence of CTP, the last 20 C-terminal residues of TarD_Sa are not visible and this section might adopt a conformation different from that observed in TagD_Bs in the presence of substrates. Detailed analysis will be presented.