Structure of Mycobacterium tuberculosis phosphatidylinositol phosphate synthase reveals mechanism of substrate binding and metal catalysis

Abstract

Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. Mycobacterium tuberculosis phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the mycobacterial cell wall, and a potential drug target. Here we present three crystal structures of M. tuberculosis PgsA1: in absence of substrates (2.9 Å), in complex with Mn 2+ and citrate (1.9 Å), and with the CDP-DAG substrate (1.8 Å). The structures reveal atomic details of substrate binding as well as coordination and dynamics of the catalytic metal site. In addition, molecular docking supported by mutagenesis indicate a binding mode for the second substrate, D- myo -inositol-3-phosphate. Together, the data describe the structural basis for M. tuberculosis phosphatidylinositol phosphate synthesis and suggest a refined general catalytic mechanism—including a substrate-induced carboxylate shift—for Class I CDP-alcohol phosphotransferases, enzymes essential for phospholipid biosynthesis in all domains of life.

The phosphatidylglycerol phosphate synthase PgsA utilizes a trifurcated amphipathic cavity for catalysis at the membrane-cytosol interface

“…They contain a strictly conserved motif (D1xxD2G1xxAR…G2xxxD3xxxD4) that is crucial for catalysis (Supplementary Fig. 2A and B) [34][35][36][37][38]. As proposed previously [36], CDP-OH_P_transf superfamily can be further classified into three families (A, B and C) basing on membrane topology and evolutionary relationship (Supplementary Fig.…”

“…The amino-terminal region of SaPgsA is fairly short with only two residues. In comparison, the structures of phosphatidylglycerol phosphate synthase (PIPS) from Renibacterium salmoninarum and Mycobacterium tuberculosis possess much longer N-terminal regions forming a juxta-membrane helix [36,37]. On the extracellular side, all six TMs of SaPgsA are positioned underneath the membrane surface (Figure 1B), creating a concave surface which may induce local membrane deformation (Figure 1D).…”

Inositol lipid synthesis is widespread in host-associated Bacteroidetes

“…The majority of Bacteroidetes encode one or the other of these pathways, indicating that inositol lipid production is a fundamental trait in the phylum. Together with the importance of inositol lipids in pathogen-host interactions 60 , their high prevalence in the host-associated Bacteroidetes suggests an unexplored role in host interactions, potentially mediated both directly through provisioning and indirectly via effects on the capsule.…”

Antibiotics and resistance: the two-sided coin of the mycobacterial cell wall

“…This occurs by way of a two-part process: PgsA1 (Rv2612c), a phosphatidylinositol phosphate synthase, initiates the biosynthesis of PI by catalysing the conjugation of D- myo -inositol-3-phosphate with cytidine diphosphate diacylglycerol ( Jackson et al, 2000 , Morii et al, 2010 ). The phosphatidylinositol phosphate formed in this reaction is then broken down into phosphatidylinositol and phosphate by an unknown phosphatase ( Grāve et al, 2019 , Jackson et al, 2000 , Morii et al, 2010 ).…”