Structural Basis for Phosphatidylinositol-Phosphate Biosynthesis

Abstract

Phosphatidylinositol is critical for intracellular signaling and anchoring of carbohydrates and proteins to outer cellular membranes. In eukaryotes, phosphatidylinositol-based lipids play important roles in numerous aspects of signal transduction and in the anchoring of glycosylphosphatidylinositol (GPI) linked proteins to the membrane. In prokaryotes, phosphatidylinositol (PI) is produced by mycobacteria, as well as some other bacterial genera, where it is required for the biosynthesis of key components of the cell wall, such as the glycolipids lipomannan and lipoarabinomannan, which are tethered to the membrane via a common PI anchor. In Mycobacterium tuberculosis, these glycolipids function as important virulence factors and modulators of the host immune response.The defining step in phosphatidylinositol biosynthesis is catalyzed by CDP-alcohol phosphotransferases (CD-APs), 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. In prokaryotes, this reaction is catalyzed by the CDP-AP phosphatidylinositol-phosphate synthase (PIPS) to yield phosphatidylinositol-phosphate, which is in turn dephosphorylated by an as yet uncharacterized enzyme to PI. Given its essentiality in mycobacterial viability—conditional knockouts prove fatal—and its divergence from the eukaryotic counterpart, PIPS can be considered a promising target for anti-tuberculosis drugs.We have determined structures of PIPS from Renibacterium salmoninarum, with and without bound CDP-diacylglycerol to 3.6 and 2.5A resolution, respectively. These structures reveal the location of the acceptor site, and the molecular determinants of substrate specificity and catalysis. Functional characterization and mutagenesis of the 40%-identical PIPS orthologfrom Mycobacterium tuberculosis 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.