A novel biosynthetic pathway of archaetidyl-myo-inositol via archaetidyl-myo-inositol phosphate from CDP-archaeol and D-glucose 6-phosphate in methanoarchaeon Methanothermobacter thermautotrophicus cells

Abstract

Ether-type inositol phospholipids are ubiquitously distributed in Archaea membranes. The present paper describes a novel biosynthetic pathway of the archaeal inositol phospholipid. To study the biosynthesis of archaetidylinositol in vitro, we prepared two possible substrates: CDP-archaeol, which was chemically synthesized, and myo-[(14)C]inositol 1-phosphate, which was enzymatically prepared from [(14)C]glucose 6-phosphate with the inositol 1-phosphate (IP) synthase of this organism. The complete structure of the IP synthase reaction product was determined to be 1l-myo-inositol 1-phosphate, based on gas liquid chromatography with a chiral column. When the two substrates were incubated with the Methanothermobacter thermautotrophicus membrane fraction, archaetidylinositol phosphate (AIP) was formed along with a small amount of archaetidylinositol (AI). The two products were identified by fast atom bombardment-mass spectrometry and chemical analyses. AI was formed from AIP by incubation with the membrane fraction, but AIP was not formed from AI. This finding indicates that archaeal AI was synthesized from CDP-archaeol and d-glucose 6-phosphate via myo-inositol 1-phosphate and AIP. Although the relevant enzymes were not isolated, three enzymes are implied: IP synthase, AIP synthase, and AIP phosphatase. AIP synthase was homologous to yeast phosphatidylinositol synthase, and we confirmed AIP synthase activity by cloning the encoding gene (MTH1691) and expressing it in Escherichia coli. AIP synthase is a newly found member of the enzyme superfamily CDP-alcohol phosphatidyltransferase, which includes a wide range of enzymes that attach polar head groups to ester- and ether-type phospholipids of bacterial and archaeal origin. This is the first report of the biosynthesis of ether-type inositol phospholipids in Archaea.

FIGURE 1.

Proposed biosynthetic pathway of AI in M. thermautotrophicus.

FIGURE 2.

Gas liquid chromatograms of trimethylsilyl-methyl derivatives of IP synthase reaction products on a cp-Chirasil-l-Val capillary column. Shown are standard mixtures of 1d- and 1l-myo-inositol 1-phosphate (A), IP synthase reaction products (B), and a IP synthase reaction mixture without M. thermautotrophicus homogenates (C).

FIGURE 3.

Analysis of the reaction products of AIP synthase. A, shown is a thin-layer chromatogram of ¹⁴C-labeled products of the AIP synthase reaction. The source of AIP synthase preparation was the membrane fraction of M. thermautotrophicus cell homogenates (lanes 1 and 2) or homogenates of E. coli pET21a-MTH1691 (lanes 3 and 4) in the presence of 0.5% Triton X-100 (lanes 1 and 3) or 0.1% Triton X-100 (lanes 2 and 4). The products were extracted and developed on TLC. Radioactive spots were detected by autoradiography. s.f., solvent front. B, shown is time-course of AIP synthase reaction. AIP and AI were formed from “[¹⁴C]inositol 1-phosphate” and CDP-archaeol catalyzed by the membrane fraction of M. thermautotrophicus cell homogenates. The reaction was stopped at the indicated time points by the addition of HCl, chloroform-soluble products were separated by TLC, and radioactivity of each spot was counted. The ratio of AIP and AI was determined by autoradiography after TLC development of the chloroform-soluble products. C and D, shown are negative ion FAB-mass spectra of AIP (C) and AI (D) enzymatically synthesized from CDP-archaeol and “myo-inositol 1-phosphate” (non-radiolabeled).

FIGURE 4.

Production of the MTH1691 gene product in E. coli. Soluble (S) and insoluble (P) fractions of E. coli cells in which the MTH1691gene was expressed were subjected to 15% SDS-PAGE, and the proteins were stained by Coomassie Brilliant Blue. The lanes shown as IPTG(−) include cell extracts from the culture without the addition of IPTG as a negative control of the gene expression. Lane M contains size marker proteins (Protein Marker, Broad range, New England Biolabs).

FIGURE 5.

Conversion of AIP to AI by the membrane fraction of M. thermautotrophicus cell homogenates or homogenates of E. coli pET21a-MTH1691. [¹⁴C]AIP and [¹⁴C]AI were prepared from CDP-archaeol and “[¹⁴C]inositol 1-phosphate” under the conditions described under “Experimental Procedures.” The purified [¹⁴C]AIP (lanes 1, 2, 3, 5, 6, 7, and 8) or [¹⁴C]AI (lane 4) was incubated at 60 °C for 2 h with either enzyme preparation (500 μg protein/30–80 μl), the membranes of M. thermautotrophicus homogenate (lanes 1, 2, and 4), the supernatant fraction of M. thermautotrophicus homogenates (lane 3), the homogenates of E. coli pET21a-MTH1691 (lanes 5 and 6), or the homogenates of E. coli pET21a carrying an empty vector plasmid (lanes 7 and 8) in the presence of 0.1% Triton X-100. Other constituents in the reaction mixture were the same as in the AIP synthase reaction. The enzyme preparation was dissolved in 50 mm phosphate buffer (lanes 1, 4, 5, and 7), in aqueous dithiothreitol solution (lanes 2 and 3), and in Bicine buffer (lanes 6 and 8). After the reaction, lipids were extracted, separated by TLC, and recorded by autoradiography. s.f., solvent front.