Publications

• Structural basis for phosphatidylinositol-phosphate biosynthesis.

  Clarke O.B., Tomasek D., Jorge C.D., Dufrisne M.B., Kim M., Banerjee S., Rajashankar K.R., Shapiro L., Hendrickson W.A., Santos H., Mancia F.

  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 ... >> More

  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. << Less

  Nat. Commun. 6:8505-8505(2015) [PubMed] [EuropePMC]

• A revised biosynthetic pathway for phosphatidylinositol in Mycobacteria.

  Morii H., Ogawa M., Fukuda K., Taniguchi H., Koga Y.

  For the last decade, it has been believed that phosphatidylinositol (PI) in mycobacteria is synthesized from free inositol and CDP-diacylglycerol by PI synthase in the presence of ATP. The role of ATP in this process, however, is not understood. Additionally, the PI synthase activity is extremely ... >> More

  For the last decade, it has been believed that phosphatidylinositol (PI) in mycobacteria is synthesized from free inositol and CDP-diacylglycerol by PI synthase in the presence of ATP. The role of ATP in this process, however, is not understood. Additionally, the PI synthase activity is extremely low compared with the PI synthase activity of yeast. When CDP-diacylglycerol and [(14)C]1L-myo-inositol 1-phosphate were incubated with the cell wall components of Mycobacterium smegmatis, both phosphatidylinositol phosphate (PIP) and PI were formed, as identified by fast atom bombardment-mass spectrometry and thin-layer chromatography. PI was formed from PIP by incubation with the cell wall components. Thus, mycobacterial PI was synthesized from CDP-diacylglycerol and myo-inositol 1-phosphate via PIP, which was dephosphorylated to PI. The gene-encoding PIP synthase from four species of mycobacteria was cloned and expressed in Escherichia coli, and PIP synthase activity was confirmed. A very low, but significant level of free [(3)H]inositol was incorporated into PI in mycobacterial cell wall preparations, but not in recombinant E. coli cell homogenates. This activity could be explained by the presence of two minor PI metabolic pathways: PI/inositol exchange reaction and phosphorylation of inositol by ATP prior to entering the PIP synthase pathway. << Less

  J. Biochem. 148:593-602(2010) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

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

  Grave K., Bennett M.D., Hoegbom M.

  Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. <i>Mycobacterium tuberculosis</i> phosphatidylinositol phosphate synthase (PgsA1) is an integral membrane enzyme involved in biosynthesis of inositol-derived phospholipids required for formation of the m ... >> More

  Tuberculosis causes over one million yearly deaths, and drug resistance is rapidly developing. <i>Mycobacterium tuberculosis</i> 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 <i>M. tuberculosis</i> PgsA1: in absence of substrates (2.9 Å), in complex with Mn²⁺ 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-<i>myo</i>-inositol-3-phosphate. Together, the data describe the structural basis for <i>M. tuberculosis</i> 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. << Less

  Commun. Biol. 2:175-175(2019) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Studies of inositol 1-phosphate analogues as inhibitors of the phosphatidylinositol phosphate synthase in mycobacteria.

  Morii H., Okauchi T., Nomiya H., Ogawa M., Fukuda K., Taniguchi H.

  We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the deve ... >> More

  We previously reported a novel pathway for the biosynthesis of phosphatidylinositol in mycobacteria via phosphatidylinositol phosphate (PIP) [Morii H., Ogawa, M., Fukuda, K., Taniguchi, H., and Koga, Y (2010) J. Biochem. 148, 593-602]. PIP synthase in the pathway is a promising target for the development of new anti-mycobacterium drugs. In the present study, we evaluated the characteristics of the PIP synthase of Mycobacterium tuberculosis. Four types of compounds were chemically synthesized based on the assumption that structural homologues of inositol 1-phosphate, a PIP synthase substrate, would act as PIP synthase inhibitors, and the results confirmed that all synthesized compounds inhibited PIP synthase activity. The phosphonate analogue of inositol 1-phosphate (Ino-C-P) had the greatest inhibitory effect among the synthesized compounds examined. Kinetic analysis indicated that Ino-C-P acted as a competitive inhibitor of inositol 1-phosphate. The IC(50) value for Ino-C-P inhibition of the PIP synthase activity was estimated to be 2.0 mM. Interestingly, Ino-C-P was utilized in the same manner as the normal PIP synthase substrate, leading to the synthesis of a phosphonate analogue of PIP (PI-C-P), which had a structure similar to that of the natural product, PIP. In addition, PI-C-P had high inhibitory activity against PIP synthase. << Less

  J. Biochem. 153:257-266(2013) [PubMed] [EuropePMC]