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Glycobiology Advance Access published online on March 20, 2006
Glycobiology, doi:10.1093/glycob/cwj104
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© The Author 2006. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org
Received February 10, 2006
Revised March 14, 2006
Accepted March 17, 2006

Article

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

Ejvis Lamani 1, R. Brandon Mewbourne 2, Damona S. Fletcher 2, Sergei D. Maltsev 3, Leonid L. Danilov 3, Vladimir V. Veselovsky 3, Antonina V. Lozanova 3, Natalia Ya. Grigorieva 3, Olga A. Pinsker 3, Jun Xing 4, W. Thomas Forsee 5, Herbert C. Cheung 4, John S. Schutzbach 5, Vladimir N. Shibaev 3, and Mark J. Jedrzejas 1 *

1 Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA
2 Children’s Hospital Oakland Research Institute, Oakland, CA 94609, USA; Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.
3 N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, 117913 Moscow, Russia
4 Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.
5 Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, U.S.A.

* To whom correspondence should be addressed.
Mark J. Jedrzejas, E-mail: mjedrzejas{at}chori.org


  Abstract

Dolichyl-Phosphate-Mannose synthase catalyzes the reversible formation of a key intermediate (Dol-P-Man) that is involved as a mannosyl donor in at least 3 different pathways for the synthesis of glycoconjugates important for eukaryotic development and viability. The enzyme is found associated with membranes of the endoplasmic reticulum where it transfers mannose from the water soluble cytoplasmic donor, GDP-Man, to the membrane-bound, extremely hydrophobic, and long chain polyisoprenoid acceptor, dolichyl-P (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 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.

Keywords: dolichyl-P/GDP-mannose/membranes/molecular mechanism of action/modeling/protein glycosylation.
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