3BUI

Golgi mannosidase II D204A catalytic nucleophile mutant complex with Tris

Summary for 3BUI

• Entry DOI: 10.2210/pdb3bui/pdb
• Related: 1HTY 1HWW 1HXK 1PS2 1QWN 1QX1 1R33 1R34 1TQS 1TQT 1TQU 1TQV 1TQW 2ALW 2F18 2F1A 2F1B 2F7O 2F7P 2F7Q 2F7R 3BUB 3BUD
• Descriptor: Alpha-mannosidase 2, ZINC ION, 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL, ... (5 entities in total)
• Functional Keywords: glycosyl hydrolase family 38, glycosidase, golgi apparatus, membrane, signal-anchor, transmembrane, hydrolase
• Biological source: Drosophila melanogaster (Fruit fly)
• Total number of polymer chains: 1
• Total formula weight: 120317.85

Authors

Kuntz, D.A.,Rose, D.R. (deposition date: 2008-01-02, release date: 2008-07-01, Last modification date: 2024-10-30)

Primary citation

Zhong, W.,Kuntz, D.A.,Ember, B.,Singh, H.,Moremen, K.W.,Rose, D.R.,Boons, G.J.
Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.
J.Am.Chem.Soc., 130:8975-8983, 2008

PubMed Abstract: 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.

PubMed: 18558690
DOI: 10.1021/ja711248y

Experimental method

X-RAY DIFFRACTION (1.25 Å)