A thermostable dolichol phosphoryl mannose synthase responsible for glycoconjugate synthesis of the hyperthermophilic archaeon Pyrococcus horikoshii

Abstract

Dolichol phosphoryl mannose synthase (DPM synthase) is an essential enzyme in the synthesis of N- and O-linked glycoproteins and the glycosylphosphatidyl-inositol anchor. An open reading frame, PH0051, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes a DPM synthase ortholog, PH0051p. A full-length version of PH0051p was produced using an E. coli in vitro translation system and its thermostable activity was confirmed with a DPM synthesis assay, although the in vitro productivity was not sufficient for further characterization. Then, a yeast expression vector coding for the N-terminal catalytic domain of PH0051p was constructed. The N-terminal domain, named DPM(1-237), was successfully expressed, and turned out to be a membrane-bound form in Saccharomyces cerevisiae cells, even without its hydrophobic C-terminal domain. The membrane-bound DPM(1-237) was solubilized with a detergent and purified to homogeneity. The purified DPM(1-237) showed thermostability at up to 75°C and an optimum temperature of 60°C. The truncated mutant DPM(1-237) required Mg²⁺ and Mn²⁺ ions as cofactors the same as eukaryotic DPM synthases. By site-directed mutagenesis, Asp⁸⁹ and Asp⁹¹ located at the most conserved motif, DXD, were confirmed as the catalytic residues, the latter probably bound to a cofactor, Mg²⁺. DPM(1-237) was able to utilize both acceptor lipids, dolichol phosphate and the prokaryotic carrier lipid C₅₅-undecaprenyl phosphate, with Km values of 1.17 and 0.59 μM, respectively. The DPM synthase PH0051p seems to be a key component of the pathway supplying various lipid-linked phosphate sugars, since P. horikoshii could synthesize glycoproteins as well as the membrane-associated PH0051p in vivo.