In Vitro Biosynthesis of Ether-Type Glycolipids in the Methanoarchaeon Methanothermobacter thermautotrophicus

Abstract

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 β- d -glucosyl-(1→6)- d -glucosyl (gentiobiosyl) unit. The enzyme activities of archaeol:UDP-glucose β-glucosyltransferase (monoglucosylarchaeol [MGA] synthase) and MGA:UDP-glucose β-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.