Novel drugs possessing a mechanism of action specific to pathogenic mycobacteria, including
Mycobacterium tuberculosis, are needed. In 2010, we discovered that the biosynthetic pathway of phosphatidylinositol, which is a membrane phospholipid, differs between humans and mycobacteria. The key enzyme responsible for this difference is phosphatidylinositol phosphate (PIP) synthase, which is present only in a few bacteria belonging to the phylum Actinobacteria. Discovering compounds that inhibit the activity of this enzyme will lead to the development of new drugs specific to pathogenic mycobacteria. Measuring PIP synthase activity requires the isotope-labeled substrate 1
l-
myo-inositol 1-phosphate (1
l-Ino-1P). Because this substrate is not commercially available, we synthesized it from [
14C] glucose 6-phosphate ([
14C] Glc-6P), using a crude enzyme solution isolated from the methanoarchaeon 1
l-Ino-1P synthase. The activity of 1
l-Ino-1P synthase in the crude enzyme mixture was low, and quantitative analysis of the synthesized 1
l-Ino-1P was inaccurate due to impurities present in the crude enzyme mixture. In the present study, we describe a method for synthesizing 1
l-Ino-1P using a solution containing recombinant 1
l-Ino-1P synthase derived from the hyperthermophilic archaeon
Aeropyrum pernix. In addition, we elucidate the conditions leading to the almost complete conversion of Glc-6P into 1
l-Ino-1P using this enzyme. Quantitation of the synthesized 1
l -Ino-1P was performed by colorimetry and gas liquid chromatography. Further, we confirmed that isotope-labeled 1
l-Ino-1P, which is difficult to quantitate by gas liquid chromatography, can be accurately quantified by colorimetry. We also confirmed that 1
d-inositol 1-phosphate cannot be a substrate for PIP synthase.
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