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Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry
08:07

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Published on: July 26, 2019

A revised biosynthetic pathway for phosphatidylinositol in Mycobacteria

Hiroyuki Morii1, Midori Ogawa, Kazumasa Fukuda

  • 1Department of Chemistry, University of Occupational and Environmental Health, Japan, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu, 807-8555, Japan. h-morii@health.uoeh-u.ac.jp

Journal of Biochemistry
|August 28, 2010
PubMed

Insights

Mycobacterial phosphatidylinositol (PI) is synthesized via phosphatidylinositol phosphate (PIP), not directly from inositol. This study identifies PIP synthase as key in mycobacterial PI production.

Area of Science:

  • Biochemistry
  • Microbiology

Background:

  • Phosphatidylinositol (PI) synthesis in mycobacteria was previously thought to involve free inositol and CDP-diacylglycerol.
  • The role of ATP and the low activity of PI synthase in mycobacteria remained unclear.
  • Enzyme activity in mycobacteria is significantly lower than in yeast.

Purpose of the Study:

  • To elucidate the mechanism of phosphatidylinositol (PI) synthesis in mycobacteria.
  • To identify the specific pathway and enzymes involved in mycobacterial PI production.
  • To investigate the role of myo-inositol 1-phosphate in PI synthesis.

Main Methods:

  • Incubation of CDP-diacylglycerol and [(14)C]1L-myo-inositol 1-phosphate with Mycobacterium smegmatis cell wall components.
  • Identification of synthesized lipids using fast atom bombardment-mass spectrometry and thin-layer chromatography.
  • Cloning and expression of the gene encoding PIP synthase from four mycobacterial species in Escherichia coli.

Main Results:

  • Mycobacterial PI is synthesized from CDP-diacylglycerol and myo-inositol 1-phosphate via phosphatidylinositol phosphate (PIP).
  • PI is formed by the dephosphorylation of PIP.
  • The gene encoding PIP synthase was successfully cloned and expressed, confirming its activity.

Conclusions:

  • Mycobacterial PI synthesis proceeds through a PIP intermediate, which is then dephosphorylated.
  • PIP synthase is a key enzyme in mycobacterial PI production.
  • Minor pathways involving PI/inositol exchange or inositol phosphorylation may also contribute to PI metabolism.

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