Dolichyl-phosphomannose synthase from the archae Thermoplasma acidophilum.
Archae (formerly Archaebacteria) comprise an entire kingdom of organisms placed halfway between prokaryotes and eucaryotes in evolution. This class of organisms lacks murein cell wall and is devoid of organelles, yet Archae synthesize and export N-linked and O-linked glycoproteins utilizing only the plasma membrane. Study of glycosylation systems in Archae is extremely interesting because the plasma membrane must perform many functions normally carried out by the endoplasmic reticulum and Golgi in eucaryotes. This report represents the first glycosyl transferase system enzyme demonstrated from archae showing a functional relationship with homologous eucaryotic enzymes. Archae dolichyl-phosphoryl-mannose synthase was purified 1070-fold from Thermoplasma acidophilum by column chromatography on Sephacryl S-200, Cibacron blue 3GA-agarose, Octyl-Sepharose, and hydroxylapatite in the presence of 0.2% polioxyethylene 9 lauryl ether. The enzyme activity was stimulated by MgCl2 (20 mM optimum) and exhibited a pH optimum at 6.0. Although the native polyisoprenoid has not been isolated or characterized, the enzyme prefers dolichyl phosphate (dol-P) to C55-polyisoprenol as an acceptor, and the Km value for dol-P was calculated to be 2.6 microM. Amphomycin, an inhibitor of dol-P-Man synthase, blocked mannosyl transfer to the endogenous lipids, proteins, and to dol-P; 100 micrograms/ml amphomycin inhibited 97% of mannosyl transfer to dol-P, and 50% to endogenous acceptors, indicating direct transfer from GDP-mannose to some intermediates or final structures. The size range of [3H]Man-oligosaccharides from acid-labile manno-lipid product was from dp 1 to 4. dol-P-Man synthase activity could be correlated directly with a 42 kDa band on SDS/polyacrylamide gel electrophoresis.
Zhu
B C
BC
Department of Biochemistry, Louisiana State University, Baton Rouge, USA.
Laine
R A
RA
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D008364:Mannosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D013822:Thermoplasma / Q000201:enzymology*
D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase