FIG 5.

Scheme representing the method used to characterize the stereospecificity of GCTs. Step 1 represents production of CDP-glycerol from rac-glycerol phosphate by the target glycerol phosphate cytidylyltransferase. Step 2 represents conversion of CDP-glycerol produced in step 1 into GPI via de action of the DIPP synthase and phosphatase activities present in the A. fulgidus cell extract provided. Finally, ³¹P NMR analysis of the reaction products enables the determination of the stereoforms of GPI produced and, indirectly, the proportions of sn-glycerol 3-phosphate and sn-glycerol 1-phosphate used by the GCT enzyme. GCT, glycerol phosphate cytidylyltransferase; DIPPS, di-myo-inositol phosphate-phosphate synthase; DIPPP, DIPP phosphatase; rac-glyP, rac-glycerol phosphate; l-inos-1P, l-myo-inositol 1-phosphate.