Investigations of the Active Site of Saccharomyces cerevisiae Dolichyl-Phosphate-Mannose Synthase Using Fluorescent Labeled Dolichyl-Phosphate Derivatives

• Published in: Biochemistry (Easton) 2000-07, Vol.39 (27), p.7886-7894
• Main Authors: Xing, Jun, Forsee, W. Thomas, Lamani, Ejvis, Maltsev, Sergei D, Danilov, Leonid L, Shibaev, Vladimir N, Schutzbach, John S, Cheung, Herbert C, Jedrzejas, Mark J
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Binding Sites; Energy Transfer; Fluorescent Dyes - chemical synthesis; Fluorescent Dyes - chemistry; Humans; Kinetics; Mannosyltransferases - chemistry; Molecular Sequence Data; Saccharomyces cerevisiae - enzymology; Sequence Homology, Amino Acid; Spectrometry, Fluorescence
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi0003240

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.