Glycobiology Advance Access originally published online on March 20, 2006
Glycobiology 2006 16(7):666-678; doi:10.1093/glycob/cwj104
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Structural studies and mechanism of Saccharomyces cerevisiae dolichyl-phosphate-mannose synthase: insights into the initial step of synthesis of dolichyl-phosphate-linked oligosaccharide chains in membranes of endoplasmic reticulum
2 Children’s Hospital Oakland Research Institute, Oakland, CA 94609; 3 Department of Microbiology, and 4Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294; and 5 N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 117913 Moscow, Russia
1 To whom correspondence should be addressed; e-mail: mjedrzejas{at}chori.org
Received on February 10, 2006; revised on March 14, 2006; accepted on March 17, 2006
Dolichyl-phosphate-mannose (Dol-P-Man) synthase catalyzes the reversible formation of a key intermediate that is involved as a mannosyl donor in at least three different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum (ER), where it transfers mannose from the water soluble cytoplasmic donor, guanosine 5'-diphosphate (GDP)-Man, to the membrane-bound, extremely hydrophobic, and long-chain polyisoprenoid acceptor, dolichyl-phosphate (Dol-P). The enzyme from Saccharomyces cerevisiae has been utilized to investigate the structure and activity of the protein and interactions of the enzyme with Dol-P and synthetic Dol-P analogs containing fluorescent probes. These interactions have been explored utilizing fluorescence resonance energy transfer (FRET) to establish intramolecular distances within the protein molecule as well as intermolecular distances to determine the localization of the active site and the hydrophobic substrate on the enzyme’s surface. A three-dimensional (3D) model of the enzyme was produced with bound substrates, Dol-P, GDP-Man, and divalent cations to delineate the binding sites for these substrates as well as the catalytic site. The FRET analysis was used to characterize the functional properties of the enzyme and to evaluate its modeled structure. The data allowed for proposing a molecular mechanism of catalysis as an inverting mechanism of mannosyl residue transfer.
Key words: dolichyl-P / GDP-mannose / membranes / molecular mechanism of action / modeling / protein glycosylation
One of the authors of this study and our friend, Professor Vladimir Nikolaevich Shibaev, unexpectedly passed away recently and we dedicate this manuscript to him and his memory.