3BVU

GOLGI MANNOSIDASE II D204A catalytic nucleophile mutant complex with Methyl(alpha-D-mannopyranosyl)-(1->3)-S-[(alpha-D-mannopyranosyl)-(1->6)]-alpha-D-mannopyranoside

Summary for 3BVU

• Entry DOI: 10.2210/pdb3bvu/pdb
• Related: 1HTY 1HWW 1HXK 1PS2 1QWN 1QWU 1QX1 1R33 1R34 1TQS 1TQT 1TQU 1TQV 1TQW 2ALW 2F18 2F1A 2F1B 2F7O 2F7P 2F7Q 2F7R 3BUB 3BUD 3BUI 3BUP 3BUQ 3BVT 3BVV 3BVW 3BVX
• Related PRD ID: PRD_900107
• Descriptor: Alpha-mannosidase 2, alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]methyl 3-thio-alpha-D-mannopyranoside, 2-acetamido-2-deoxy-beta-D-glucopyranose, ... (7 entities in total)
• Functional Keywords: family 38 glycoysl hydrolase, glycosidase, golgi apparatus, membrane, signal-anchor, transmembrane, hydrolase
• Biological source: Drosophila melanogaster (Fruit fly)
• Total number of polymer chains: 1
• Total formula weight: 120691.89

Authors

Kuntz, D.A.,Rose, D.R. (deposition date: 2008-01-07, release date: 2008-07-01, Last modification date: 2024-11-13)

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 (1.12 Å)