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.
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.
Lamani
Ejvis
E
Children's Hospital Oakland Research Institute, CA 94609, USA.
Mewbourne
R Brandon
RB
Fletcher
Damona S
DS
Maltsev
Sergei D
SD
Danilov
Leonid L
LL
Veselovsky
Vladimir V
VV
Lozanova
Antonina V
AV
Grigorieva
Natalia Ya
NY
Pinsker
Olga A
OA
Xing
Jun
J
Forsee
W Thomas
WT
Cheung
Herbert C
HC
Schutzbach
John S
JS
Shibaev
Vladimir N
VN
Jedrzejas
Mark J
MJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002384:Catalysis; D004287:Dolichol Monophosphate Mannose / Q000378:metabolism; D004288:Dolichol Phosphates / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005456:Fluorescent Dyes / Q000737:chemistry; D007425:Intracellular Membranes / Q000201:enzymology; D008364:Mannosyltransferases / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000096:biosynthesis*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D013379:Substrate Specificity
D004288:Dolichol Phosphates; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; C028306:dolichol monophosphate; D004287:Dolichol Monophosphate Mannose; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase