Phosphatidylinositol synthesis in mycobacteria

The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacteri um smegmatis and M. smegmatis subcellular fractions. Little is known about the synthesis of PI in prokaryotic cells. Only a cell wall fraction (P60) i n M. smegmatis was shown to possess PI synthase activity. Product was ident ified as PI by migration on TLC, treatment with phospholipase C and ion exc hange chromatography. PI was the only major product (92.3%) when both cells and P60 fraction were labeled with [H-3]inositol. Also, a neutral lipid in ositol-containing product (4.1% of the total label) was identified in the P 60 preparations. Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate [H-3]PI and NBD-PI yield by M. smegmatis. At the same time, addition of both substrates to rat live r and saccharomyces cerevisiae PI synthase assays resulted in an increase i n the product yield. Upon addition of CHAPS to the mycobacterial PI synthas e assay, both substrates were utilized in a dose-dependent manner for the s ynthesis of NBD-PI and [H-3]PI. These results demonstrate a strict substrat e specificity of mycobacterial PI synthase toward endogenous substrates, K- m of the enzyme toward inositol was shown to be 25 mu M: Mg2+ stimulated th e enzyme to a greater degree than Mn2+. Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inh ibitors of mycobacterial PI synthase than of mammalian analogs. Lack of seq uence homology with mammalian PI synthases, different kinetic characteristi cs, existence of selective inhibitors and an important physiological role i n mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy. (C) 1999 Elsevier Science B.V. All rights reserv ed.