Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. The defining step in phosphatidylinositol biosynthesis is catalysed by CDP-alcohol phosphotransferases, transmembrane enzymes that use CDP-diacylglycerol as donor substrate for this reaction, and either inositol in eukaryotes or inositol phosphate in prokaryotes as the acceptor alcohol. Here we report the structures of a related enzyme, the phosphatidylinositol-phosphate synthase from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5 Å resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization of the 40%-identical ortholog from Mycobacterium tuberculosis, a potential target for the development of novel anti-tuberculosis drugs, supports the proposed mechanism of substrate binding and catalysis. This work therefore provides a structural and functional framework to understand the mechanism of phosphatidylinositol-phosphate biosynthesis.

The intracellular accumulation of low molecular mass organic compounds in response to stressful conditions was investigated in the thermophilic bacterium Petrotoga miotherma, a member of the order Thermotogales. This led to the discovery of a new solute, whose structure was established as α‐d‐mannopyranosyl‐(1→2)‐α‐d‐glucopyranosyl‐(1→2)‐glycerate (MGG) by MMR spectroscopy and MS. Under optimum growth conditions (3% NaCl; 55 °C), MGG was the major solute [up to 0.6 µmol·(mg protein)⁻¹]; α‐glutamate and proline were also present but in minor amounts [below 0.08 µmol·(mg protein)⁻¹]. The level of MGG increased notably with the salinity of the growth medium up to the optimum NaCl concentration. At higher NaCl concentrations, however, the level of MGG decreased, whereas the levels of proline and α‐glutamate increased about five‐fold and 10‐fold, respectively. MGG plays a role during low‐level osmotic adaptation of Petrotoga miotherma, whereas α‐glutamate and, to a lesser extent, proline are used for osmoprotection under salt stress. MGG is not part of the cell strategy for coping with heat or oxidative stress. Nevertheless, MGG was an efficient protector of pig heart malate dehydrogenase against heat inactivation and freeze‐drying, although mannosylglycerate was better. This is the first report on the occurrence of MGG in living systems.