A thermostable dolichol phosphoryl mannose synthase responsible for glycoconjugate synthesis of the hyperthermophilic archaeon Pyrococcus horikoshii
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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.
KeywordsDolichol phosphoryl mannose synthase Membrane protein Hyperthermophilic archaea Pyrococcus Glycoprotein synthesis Glycoconjugate Glycosylphosphatidyl-inositol anchor Thermostable flippase
This work was supported in part by the science program of the New Energy and Industrial Technology Development Organization, Japan. We thank Emiko Yamamoto for technical support.
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