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|3bud, resolution 1.85Å ()|
|Ligands:||, , ,|
|Gene:||alpha-Man-II, GmII (Drosophila melanogaster)|
|Related:||1hty, 1hww, 1hxk, 1ps2, 1qwn, 1qx1, 1r33, 1r34, 1tqs, 1tqt, 1tqu, 1tqv, 1tqw, 2alw, 2f18, 2f1a, 2f1b, 2f7o, 2f7p, 2f7q, 2f7r, 3bub, 3bui|
Golgi mannosidase II D204A catalytic nucleophile mutant with an empty active site
Inhibition of Golgi alpha-mannosidase II (GMII), which acts late in the N-glycan processing pathway, provides a route to blocking cancer-induced changes in cell surface oligosaccharide structures. To probe the substrate requirements of GMII, oligosaccharides were synthesized that contained an alpha(1,3)- or alpha(1,6)-linked 1-thiomannoside. Surprisingly, these oligosaccharides were not observed in X-ray crystal structures of native Drosophila GMII (dGMII). However, a mutant enzyme in which the catalytic nucleophilic aspartate was changed to alanine (D204A) allowed visualization of soaked oligosaccharides and led to the identification of the binding site for the alpha(1,3)-linked mannoside of the natural substrate. These studies also indicate that the conformational change of the bound mannoside to a high-energy B 2,5 conformation is facilitated by steric hindrance from, and the formation of strong hydrogen bonds to, Asp204. The observation that 1-thio-linked mannosides are not well tolerated by the catalytic site of dGMII led to the synthesis of a pentasaccharide containing the alpha(1,6)-linked Man of the natural substrate and the beta(1,2)-linked GlcNAc moiety proposed to be accommodated by the extended binding site of the enzyme. A cocrystal structure of this compound with the D204A enzyme revealed the molecular interactions with the beta(1,2)-linked GlcNAc. The structure is consistent with the approximately 80-fold preference of dGMII for the cleavage of substrates containing a nonreducing beta(1,2)-linked GlcNAc. By contrast, the lysosomal mannosidase lacks an equivalent GlcNAc binding site and kinetic analysis indicates oligomannoside substrates without non-reducing-terminal GlcNAc modifications are preferred, suggesting that selective inhibitors for GMII could exploit the additional binding specificity of the GlcNAc binding site.
Probing the Substrate Specificity of Golgi alpha-Mannosidase II by Use of Synthetic Oligosaccharides and a Catalytic Nucleophile Mutant., Zhong W, Kuntz DA, Ember B, Singh H, Moremen KW, Rose DR, Boons GJ, J Am Chem Soc. 2008 Jun 18;. PMID:18558690
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.