PDBsum entry 2gl9

Hydrolase

PDB id: 2gl9

Contents

Protein chains

140 a.a. *

144 a.a. *

Ligands

NAG ×2

ASN ×2

Waters ×377

* Residue conservation analysis

PDB id:

2gl9

Name:

Hydrolase

Title:

Crystal structure of glycosylasparaginase-substrate complex

Structure:

Glycosylasparaginase alpha chain. Chain: a, c. Fragment: residues 46-196. Engineered: yes. Glycosylasparaginase beta chain. Chain: b, d. Fragment: residues 197-340. Engineered: yes. Mutation: yes

Source:

Elizabethkingia meningoseptica. Organism_taxid: 238. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.00Å R-factor: 0.170 R-free: 0.215

Authors:

Y.Wang,H.C.Guo

Key ref:

Y.Wang and H.C.Guo (2007). Crystallographic snapshot of a productive glycosylasparaginase-substrate complex. J Mol Biol, 366, 82-92. PubMed id: 17157318 DOI: 10.1016/j.jmb.2006.09.051

Date:

04-Apr-06 Release date: 13-Feb-07

Protein chains

Q47898  (ASPG_ELIMR) -  N(4)-(Beta-N-acetylglucosaminyl)-L-asparaginase from Elizabethkingia miricola

Seq: 340 a.a.

Struc: 140 a.a.

Protein chains

Q47898  (ASPG_ELIMR) -  N(4)-(Beta-N-acetylglucosaminyl)-L-asparaginase from Elizabethkingia miricola

Seq: 340 a.a.

Struc: 144 a.a.*

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

Enzyme reactions

• Enzyme class: Chains A, B, C, D: E.C.3.5.1.26 - N(4)-(beta-N-acetylglucosaminyl)-L-asparaginase.
• Reaction: N₄-(beta-N-acetyl-D-glucosaminyl)-L-asparagine + H2O = N-acetyl-beta-D- glucosaminylamine + L-aspartate + H⁺

N(4)-(beta-N-acetyl-D-glucosaminyl)-L-asparagine + H2O = N-acetyl-beta-D- glucosaminylamine + L-aspartate (Bound ligand (Het Group name = ASN) matches with 80.00% similarity) + H(+) (Bound ligand (Het Group name = NAG) matches with 93.33% similarity)

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

reference

DOI no: 10.1016/j.jmb.2006.09.051

J Mol Biol 366:82-92 (2007)

PubMed id: 17157318

Crystallographic snapshot of a productive glycosylasparaginase-substrate complex.

Y.Wang, H.C.Guo.

ABSTRACT

Glycosylasparaginase (GA) plays an important role in asparagine-linked glycoprotein degradation. A deficiency in the activity of human GA leads to a lysosomal storage disease named aspartylglycosaminuria. GA belongs to a superfamily of N-terminal nucleophile hydrolases that autoproteolytically generate their mature enzymes from inactive single chain protein precursors. The side-chain of the newly exposed N-terminal residue then acts as a nucleophile during substrate hydrolysis. By taking advantage of mutant enzyme of Flavobacterium meningosepticum GA with reduced enzymatic activity, we have obtained a crystallographic snapshot of a productive complex with its substrate (NAcGlc-Asn), at 2.0 A resolution. This complex structure provided us an excellent model for the Michaelis complex to examine the specific contacts critical for substrate binding and catalysis. Substrate binding induces a conformational change near the active site of GA. To initiate catalysis, the side-chain of the N-terminal Thr152 is polarized by the free alpha-amino group on the same residue, mediated by the side-chain hydroxyl group of Thr170. Cleavage of the amide bond is then accomplished by a nucleophilic attack at the carbonyl carbon of the amide linkage in the substrate, leading to the formation of an acyl-enzyme intermediate through a negatively charged tetrahedral transition state.

Selected figure(s)

Figure 1.
Figure 1. Hydrolysis reaction catalyzed by GA amidase. GA cleaves the β-N-aspartylglucosylamine bond (indicated by the bold arrow) of its natural substrate NAcGlc-Asn during proteolytic processings of asparagine-linked glycoproteins, resulting in the release of apartic acid and aminoglycan. The latter product is then further hydrolyzed non-enzymatically to release ammonia and oligosaccharide. Figure 1. Hydrolysis reaction catalyzed by GA amidase. GA cleaves the β-N-aspartylglucosylamine bond (indicated by the bold arrow) of its natural substrate NAcGlc-Asn during proteolytic processings of asparagine-linked glycoproteins, resulting in the release of apartic acid and aminoglycan. The latter product is then further hydrolyzed non-enzymatically to release ammonia and oligosaccharide.

Figure 4.
Figure 4. Stereo view of the atomic interactions between GA and substrate. Displayed are interactions between GA and the bound substrate molecule NAcGlc-Asn at the active site A. The active side-chain conformation of residue Cys152 is shown in magenta and the switch between its inactive trans- and active gauche(+) conformations is indicated by the magenta double arrow. Nucleophilic attack is indicated by the green straight arrow. A candidate water molecule to protonate the leaving group is also shown (W). The green dotted lines indicate possible hydrogen-bonding interactions between Cys152 and the surrounding residues. The blue dotted lines denote other hydrogen bonds involved in enzyme–substrate binding. Also shown is a hydrogen bond (a black dotted line) between side-chains of Trp11 and Thr203. Key active site residues are shown by atom type: yellow for carbon, blue for nitrogen, red for oxygen, and green for the Cys152 sulfur atom. The salt bridge is indicated by the positive and the negative charges. Figure 4. Stereo view of the atomic interactions between GA and substrate. Displayed are interactions between GA and the bound substrate molecule NAcGlc-Asn at the active site A. The active side-chain conformation of residue Cys152 is shown in magenta and the switch between its inactive trans- and active gauche(+) conformations is indicated by the magenta double arrow. Nucleophilic attack is indicated by the green straight arrow. A candidate water molecule to protonate the leaving group is also shown (W). The green dotted lines indicate possible hydrogen-bonding interactions between Cys152 and the surrounding residues. The blue dotted lines denote other hydrogen bonds involved in enzyme–substrate binding. Also shown is a hydrogen bond (a black dotted line) between side-chains of Trp11 and Thr203. Key active site residues are shown by atom type: yellow for carbon, blue for nitrogen, red for oxygen, and green for the Cys152 sulfur atom. The salt bridge is indicated by the positive and the negative charges.

The above figures are reprinted from an Open Access publication published by Elsevier: J Mol Biol (2007, 366, 82-92) copyright 2007.

Figures were selected by an automated process.