PDBsum entry 1x9d

Hydrolase

PDB id: 1x9d

Contents

Protein chain

452 a.a. *

Ligands

Z5L-MAN

SO4

BU1

Metals

_CA

Waters ×376

* Residue conservation analysis

PDB id:

1x9d

Name:

Hydrolase

Title:

Crystal structure of human class i alpha-1,2-mannosidase in complex with thio-disaccharide substrate analogue

Structure:

Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha- mannosidase. Chain: a. Fragment: residues 243-699. Synonym: er alpha-1,2-mannosidase, mannosidase alpha class 1b member 1, man9glcnac2-specific processing alpha-mannosidase, unq747/pro1477. Engineered: yes

Source:

Homo sapiens. Human. Organism_taxid: 9606. Gene: man1b1. Expressed in: pichia pastoris. Expression_system_taxid: 4922

Resolution:

1.41Å R-factor: 0.146 R-free: 0.162

Authors:

K.Karaveg,W.Tempel,Z.J.Liu,A.Siriwardena,K.W.Moremen,B.C.Wang

Key ref:

K.Karaveg et al. (2005). Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control. J Biol Chem, 280, 16197-16207. PubMed id: 15713668 DOI: 10.1074/jbc.M500119200

Date:

20-Aug-04 Release date: 22-Feb-05

Protein chain

Q9UKM7  (MA1B1_HUMAN) -  Endoplasmic reticulum mannosyl-oligosaccharide 1,2-alpha-mannosidase from Homo sapiens

Seq: 699 a.a.

Struc: 452 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.113 - mannosyl-oligosaccharide 1,2-alpha-mannosidase.
• Reaction:
  1. N₄-(alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)- [alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->2)-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] (N-glucan mannose isomer 9A1,2,3B1,2,3) + 4 H2O = N₄-(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] (N-glucan mannose isomer 5A1,2) + 4 beta-D-mannose
  2. N₄-(alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)- [alpha-D-Man-(1->3)-[alpha-D-Man-(1->2)-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] (N-glucan mannose isomer 8A1,2,3B1,3) + 3 H2O = N₄-(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] (N-glucan mannose isomer 5A1,2) + 3 beta-D-mannose.CC -!- This family of mammalian enzymes, located in the Golgi system,

N(4)-(alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)- [alpha-D-Man-(1->2)-alpha-D-Man-(1->3)-[alpha-D-Man-(1->2)-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] (N-glucan mannose isomer 9A1,2,3B1,2,3) + 4 × H2O = N(4)-(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] (N-glucan mannose isomer 5A1,2) (Bound ligand (Het Group name = Z5L) matches with 78.57% similarity) + 4 × beta-D-mannose

N(4)-(alpha-D-Man-(1->2)-alpha-D-Man-(1->2)-alpha-D-Man-(1->3)- [alpha-D-Man-(1->3)-[alpha-D-Man-(1->2)-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] (N-glucan mannose isomer 8A1,2,3B1,3) + 3 × H2O = N(4)-(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] (N-glucan mannose isomer 5A1,2) (Bound ligand (Het Group name = Z5L) matches with 78.57% similarity) + 3 × beta-D-mannose.CC -!- This family of mammalian enzymes, located in the Golgi system,

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

DOI no: 10.1074/jbc.M500119200

J Biol Chem 280:16197-16207 (2005)

PubMed id: 15713668

Mechanism of class 1 (glycosylhydrolase family 47) {alpha}-mannosidases involved in N-glycan processing and endoplasmic reticulum quality control.

K.Karaveg, A.Siriwardena, W.Tempel, Z.J.Liu, J.Glushka, B.C.Wang, K.W.Moremen.

ABSTRACT

Quality control in the endoplasmic reticulum (ER) determines the fate of newly synthesized glycoproteins toward either correct folding or disposal by ER-associated degradation. Initiation of the disposal process involves selective trimming of N-glycans attached to misfolded glycoproteins by ER alpha-mannosidase I and subsequent recognition by the ER degradation-enhancing alpha-mannosidase-like protein family of lectins, both members of glycosylhydrolase family 47. The unusual inverting hydrolytic mechanism catalyzed by members of this family is investigated here by a combination of kinetic and binding analyses of wild type and mutant forms of human ER alpha-mannosidase I as well as by structural analysis of a co-complex with an uncleaved thiodisaccharide substrate analog. These data reveal the roles of potential catalytic acid and base residues and the identification of a novel (3)S(1) sugar conformation for the bound substrate analog. The co-crystal structure described here, in combination with the (1)C(4) conformation of a previously identified co-complex with the glycone mimic, 1-deoxymannojirimycin, indicates that glycoside bond cleavage proceeds through a least motion conformational twist of a properly predisposed substrate in the -1 subsite. A novel (3)H(4) conformation is proposed as the exploded transition state.

Selected figure(s)

Figure 5.
FIG. 5. Normalized F[o] - F[c] disaccharide electron density map for the thioin the active site of ERManI and comparison of the sugar ring conformations with the enzyme-bound conformation of dMNJ in the -1 subsite and the M7 mannose residue in the +1 subsite. A, a stereographic representation of the difference electron density for the omitted inhibitor in the ERManI-thiodisaccharide cocomplex. The inhibitor model is shown to aid in map interpretation. The reducing terminal Man- -O-CH[3] is shown at the top, labeled as the +1 subsite residue, and the nonreducing terminal Man residue in the 3S[1] conformation is labeled as the -1 subsite residue. Carbon and sulfur atoms in the structures are labeled as a reference. The electron density map was contoured at 3 for the gray mesh and 10 for the red mesh, demonstrating the significant electron density at the glycosidic sulfur, the O-3' and O-4' hydroxyls of the +1 residue, and the O-2', O-3', and O-4' hydroxyls of the -1 residue. B, the protein structure of the ER-ManI-thiodisaccharide co-complex was aligned with the corresponding protein structures of the ERManI·dMNJ co-complex (20) and the co-complex of yeast ERManI containing a Man[5]GlcNAc[2] glycan in the active site (21) using Swiss-PdbViewer (version 3.7) (55). Displayed in the figure are the structures of the thiodisaccharide (yellow stick figure), dMNJ (green stick figure), and the M7 residue of the Man[5]GlcNAc[2] glycan in the +1 subsite (white stick figure; see Ref. 19 for oligosaccharide residue nomenclature). Carbon and sulfur atoms in the structures are labeled as a reference. The M7 residue is in an identical conformation as the +1 residue of the thiodisaccharide and in a similar position except for an offset of 0.5-0.7 Å resulting from the longer C-S bond lengths of the thiodisaccharide. The positioning of the -1 subsite residues (dMNJ versus the -1 residue of the thiodisaccharide) were virtually identical at the C-2, C-3, and C-4 positions. The main differences between the two structures were found in the equivalent of the C-1, O-5, C-5, and C-6 positions.

Figure 7.
FIG. 7. Interactions between the thiodisaccharide and the +1 and -1 binding sites in human ERManI. Shown is a schematic diagram (left panel) of the interactions between the thiodisaccharide and ERManI in the -1 and +1 subsites, demonstrating hydrogen bonding interactions (green dotted lines), direct coordination of the enzyme-associated Ca^2+ ion (blue dotted lines), hydrophobic stacking of Phe^659 with the C-4-C-5-C-6 region of the -1 residue (black dotted lines), and proposed acid-catalyzed through-water (W8) protonation of the glycosidic oxygen (sulfur in the thiodisaccharide) and the base-catalyzed (Glu599) attack by the water nucleophile (W5) (red dotted lines). Residue numbering of amino acid side chains in the respective subsites is indicated. The stereo view (center and right) illustrates a stick diagram of the interaction between the thiodisaccharide residues in the -1 and +1 subsites relative to the residues examined by mutagenesis here. Coordination to the Ca^2+ ion (blue dotted lines), and the proposed nucleophile trajectory and acid protonation of the glycosidic oxygen (red dotted lines) are indicated. The small red and green space fill structures representing the water molecules and carbonyl oxygen and O- of Thr688 that coordinate the Ca^2+ ion are as described in the legend to Fig. 2. The green dotted lines indicate hydrogen bonds between the respective residues.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2005, 280, 16197-16207) copyright 2005.

Figures were selected by an automated process.