Investigations of the Active Site of Saccharomyces cerevisiae Dolichyl-Phosphate-Mannose Synthase Using Fluorescent Labeled Dolichyl-Phosphate Derivatives
Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesi...
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Published in | Biochemistry (Easton) Vol. 39; no. 27; pp. 7886 - 7894 |
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Main Authors | , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Chemical Society
11.07.2000
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Subjects | |
Online Access | Get full text |
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Summary: | Dolichol-phosphate mannose (Dol-P-Man) is a key mannosyl donor for the biosynthesis of N-linked oligosaccharides as well as for O-linked oligosaccharides on yeast glycoproteins, and for the synthesis of the glycosyl-phosphatidylinositol anchor found on many cell surface glycoproteins. It is synthesized by Dol-P-Man synthase which is the only glycosyltransferase in the dolichol pathway that has been expressed as an active protein, solubilized and purified in large enough quantities for structural investigations. Earlier studies showed that the enzyme is closely associated with membranes of endoplasmic reticulum with unique lipid requirements for its maximal activity. This potential target of antibiotic therapy is now being investigated at the molecular level to establish information about the structure of the enzyme as well as determine the nature and properties of the enzyme−phospholipid interactions. In this paper, we have determined the activities of the fluorescent labeled dolichyl-phosphate derivatives as well as the intramolecular distances between amino acid residues near the active site and/or the fluorophores of the substrate derivatives using fluorescence energy resonance transfer. These results also show that the conserved consensus sequence is not required by Dol-P-Man synthase neither for the recognition of Dol-P nor for the catalytic activity. |
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Bibliography: | This work was supported by in part by U.S. Civilian Research and Development Foundation Grant RN1-404 (to M.J.J. and V.N.S.) and in part by an American Cancer Society Institutional Research Grant (to M.J.J.), and the time-resolved fluorescence instrumentation was supported by National Institutes of Health Grant RR10404 (to H.C.C.). istex:98EBFCBA05AF9663471AB01C65C6958F1774D54B ark:/67375/TPS-N408Z5S7-6 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0006-2960 1520-4995 |
DOI: | 10.1021/bi0003240 |