PDBsum entry 3bub

Hydrolase

PDB id: 3bub

Contents

Protein chain

1016 a.a. *

Ligands

NAG

MPD ×3

Metals

_ZN

Waters ×1493

* Residue conservation analysis

PDB id:

3bub

Name:

Hydrolase

Title:

Golgi alpha-mannosidase ii with an empty active site

Structure:

Alpha-mannosidase 2. Chain: a. Fragment: catalytic domain. Unp residues 76-1108. Synonym: alpha-mannosidase ii, mannosyl-oligosaccharide 1,3-1,6- alpha-mannosidase, man ii, golgi alpha-mannosidase ii, aman ii. Engineered: yes

Source:

Drosophila melanogaster. Fruit fly. Gene: alpha-man-ii, gmii. Expressed in: drosophila melanogaster.

Resolution:

1.38Å R-factor: 0.180 R-free: 0.204

Authors:

D.A.Kuntz,D.R.Rose

Key ref:

W.Zhong et al. (2008). 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. PubMed id: 18558690

Date:

02-Jan-08 Release date: 01-Jul-08

Protein chain

Q24451  (MAN2_DROME) -  Alpha-mannosidase 2 from Drosophila melanogaster

Seq: 1108 a.a.

Struc: 1016 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.2.1.114 - mannosyl-oligosaccharide 1,3-1,6-alpha-mannosidase.
• Pathway: Mannosyl-glycoprotein N-acetylglucosaminyltransferases
• Reaction: N₄-{beta-D-GlcNAc-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->3)-[alpha- D-Man-(1->6)]-alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)- beta-D-GlcNAc}-L-asparaginyl-[protein] + 2 H2O = 2 alpha-D-mannopyranose + an N₄-{beta-D-GlcNAc-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->6)]- beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-beta-D-GlcNAc}-L-asparaginyl- [protein]

J Am Chem Soc 130:8975-8983 (2008)

PubMed id: 18558690

Probing the substrate specificity of Golgi alpha-mannosidase II by use of synthetic oligosaccharides and a catalytic nucleophile mutant.

W.Zhong, D.A.Kuntz, B.Ember, H.Singh, K.W.Moremen, D.R.Rose, G.J.Boons.

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.