Structural basis for phosphatidylinositol-phosphate biosynthesis.

Oliver B Clarke,Minah Kim,Carla D Jorge,David Tomasek,Surajit Banerjee,Meagan Belcher Dufrisne,Lawrence Shapiro,Filippo Mancia,Helena Santos,Wayne A Hendrickson,Kanagalaghatta R Rajashankar

doi:10.1038/ncomms9505

Abstract

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.

Highlights

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes
Unlike most other CDP-alcohol phosphotransferases (CDP-APs), both Af2299 and AfDIPP synthase have an N-terminal cytosolic cytidylyltransferase-like domain[14,15], which provided the essential contacts in the crystal lattice between layers of molecules[8]
The defining step in glycerophospholipid biosynthesis is catalysed by CDP-APs

Summary

Introduction

Phosphatidylinositol is critical for intracellular signalling and anchoring of carbohydrates and proteins to outer cellular membranes. A homologous enzyme (archaetidyl-myo-inositol phosphate synthase) has been characterized in archaea, which like the PIP synthases uses inositol phosphate as the acceptor, but requires CDP-archaeol, an isoprene-based ether-linked lipid, as the donor instead of CDP-DAG5. Each of these enzymes is a member of the class I CDP-alcohol phosphotransferases (CDP-APs); class II CDP-APs are peripheral membrane proteins of an unrelated fold, and are not involved in PI biosynthesis (class II enzymes will not be discussed here, and we will use the term CDP-APs as referred to class I family members only). This, combined with the unique pathway used for PI synthesis in prokaryotes, may position PIP synthase as a potentially attractive future drug target[12,13]

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Results

Conclusion