In vitro biosynthesis of ether-type glycolipids in the methanoarchaeon Methanothermobacter thermautotrophicus

Abstract

The biosynthesis of archaeal ether-type glycolipids was investigated in vitro using Methanothermobacter thermautotrophicus cell-free homogenates. The sole sugar moiety of glycolipids and phosphoglycolipids of the organism is the beta-D-glucosyl-(1-->6)-D-glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose beta-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose beta-1,6-glucosyltransferase (diglucosylarchaeol [DGA] synthase) were found in the methanoarchaeon. The synthesis of DGA is probably a two-step glucosylation: (i) archaeol + UDP-glucose --> MGA + UDP, and (ii) MGA + UDP-glucose --> DGA + UDP. Both enzymes required the addition of K(+) ions and archaetidylinositol for their activities. DGA synthase was stimulated by 10 mM MgCl(2), in contrast to MGA synthase, which did not require Mg(2+). It was likely that the activities of MGA synthesis and DGA synthesis were carried out by different proteins because of the Mg(2+) requirement and their cellular localization. MGA synthase and DGA synthase can be distinguished in cell extracts greatly enriched for each activity by demonstrating the differing Mg(2+) requirements of each enzyme. MGA synthase preferred a lipid substrate with the sn-2,3 stereostructure of the glycerol backbone on which two saturated isoprenoid chains are bound at the sn-2 and sn-3 positions. A lipid substrate with unsaturated isoprenoid chains or sn-1,2-dialkylglycerol configuration exhibited low activity. Tetraether-type caldarchaetidylinositol was also actively glucosylated by the homogenates to form monoglucosyl caldarchaetidylinositol and a small amount of diglucosyl caldarchaetidylinositol. The addition of Mg(2+) increased the formation of diglucosyl caldarchaetidylinositol. This suggested that the same enzyme set synthesized the sole sugar moiety of diether-type glycolipids and tetraether-type phosphoglycolipids.

FIG. 1.

Possible pathways of β-d-glucosyl-(1→6)-β-d-glucosyl archaeol (DGA) from archaeol via β-d-glucosyl archaeol (MGA).

FIG. 2.

Time courses of MGA and DGA synthesis from archaeol (A and B) and DGA synthesis from MGA (C and D). (A and B) Synthesis of glycolipids from 100 nmol UDP-[U-¹⁴C]glucose and 20 nmol archaeol catalyzed by the supernatant fraction of M. thermautotrophicus homogenate fraction AS incubated in the presence of 0.5 M KCl and 20 nmol archaetidylinositol in a final volume of 0.1 ml at 60°C, pH 8.0. (C and D) The enzyme assay condition was similar to that of the above assay except that the membrane fraction of M. thermautotrophicus homogenate fraction BM was used as an enzyme source, the lipid substrate was replaced by 40 nmol MGA, and 10 mM MgCl₂ was added. (B and D) Autoradiograms of one-dimensional TLC of ¹⁴C products taken at indicated times (shown in minutes).

FIG. 3.

Negative ion FAB mass spectra of MGA (A) and DGA (B) enzymatically prepared from archaeol and MGA, respectively.

FIG. 4.

¹H-NMR spectra of MGA (A) and DGA (B) (500 MHz in CDCl₃ for MGA and in CDCl₃-CD₃OD (7:3) for DGA).

FIG. 5.

Effects of K⁺ concentration on MGA synthase activity and DGA synthase activity.

FIG. 6.

Effects of pH on MGA synthase activity (A) and DGA synthase activity (B).

FIG. 7.

Effects of Mg²⁺ concentration on DGA synthase activity (A) and (B and C) MGA synthase assay by use of the supernatant fraction AS (B) and the membrane fraction BM (C) of M. thermautotrophicus homogenate as an enzyme source. Glc, glucose.

FIG. 8.

Requirement of UDP-glucose for DGA synthase activity. DGA synthase reactions were carried out with [¹⁴C]MGA in the presence (left lane) or absence (right lane) of nonradioactive UDP-glucose. The products were analyzed by one-dimensional TLC. An autoradiogram of the TLC was shown.

FIG. 9.

Possible pathways of DGCI synthesis from caldarchaetidylinositol. G, glucose.