PDBsum entry: 3esw

Hydrolase

PDB id: 3esw

Contents

Protein chains

333 a.a. *

55 a.a. *

Ligands

NAG-NAG

Metals

_ZN

Waters ×7

* Residue conservation analysis

PDB id:

3esw

Name:

Hydrolase

Title:

Complex of yeast pngase with glcnac2-iac.

Structure:

Peptide-n(4)-(n-acetyl-beta-glucosaminyl)asparagi amidase. Chain: a. Fragment: peptide:n-glycanase. Synonym: pngase, peptide:n-glycanase 1, ypng1. Engineered: yes. Uv excision repair protein rad23. Chain: b. Fragment: xpcb domain.

Source:

Saccharomyces cerevisiae. Yeast. Organism_taxid: 4932. Gene: png1, ypl096w. Expressed in: escherichia coli. Expression_system_taxid: 562. Gene: rad23, sygp-orf29, yel037c.

Resolution:

3.40Å R-factor: 0.199 R-free: 0.235

Authors:

G.Zhao,X.Zhou,W.J.Lennarz,H.Schindelin

Key ref:

G.Zhao et al. (2009). Structural and mutational studies on the importance of oligosaccharide binding for the activity of yeast PNGase. Glycobiology, 19, 118-125. PubMed id: 18854368 DOI: 10.1093/glycob/cwn108

Date:

06-Oct-08 Release date: 11-Nov-08

Protein chain

Q02890  (PNG1_YEAST) -  Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase

Seq: 363 a.a.

Struc: 333 a.a.*

Protein chain

P32628  (RAD23_YEAST) -  UV excision repair protein RAD23

Seq: 398 a.a.

Struc: 55 a.a.

* PDB and UniProt seqs differ at 8 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chain A: E.C.3.5.1.52 - Peptide-N(4)-(N-acetyl-beta-glucosaminyl)asparagine amidase.
• Reaction: Hydrolysis of an N(4)-(acetyl-beta-D-glucosaminyl)asparagine residue in which the N-acetyl-D-glucosamine residue may be further glycosylated, to yield a (substituted) N-acetyl-beta-D- glucosaminylamine and the peptide containing an aspartic residue.

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (4 terms)
• Biological process: protein deglycosylation (4 terms)
• Biochemical function: protein binding (5 terms)

DOI no: 10.1093/glycob/cwn108

Glycobiology 19:118-125 (2009)

PubMed id: 18854368

Structural and mutational studies on the importance of oligosaccharide binding for the activity of yeast PNGase.

G.Zhao, G.Li, X.Zhou, I.Matsuo, Y.Ito, T.Suzuki, W.J.Lennarz, H.Schindelin.

ABSTRACT

Peptide:N-glycanase (PNGase) is an important component of the endoplasmic reticulum-associated protein degradation pathway in which it de-glycosylates misfolded glycoproteins, thus facilitating their proteasomal degradation. PNGase belongs to the transglutaminase superfamily and features a Cys, His, and Asp catalytic triad, which is essential for its enzymatic activity. An elongated substrate-binding groove centered on the active site Cys191 was visualized in the crystal structure of apo-PNGase, whereas its complex with Z-VAD-fmk, a peptide-based inhibitor of PNGase, revealed that the inhibitor occupied one end of the substrate-binding groove while being covalently linked to the active site Cys. Recently, haloacetamidyl-containing carbohydrate-based inhibitors of PNGase were developed and shown to specifically label the active site Cys. In this study, we describe the crystal structure of yeast PNGase in complex with N,N'-diacetylchitobiose (chitobiose). We found that the chitobiose binds on the side opposite to the peptide binding site with the active site Cys191 being located approximately midway between the carbohydrate and peptide binding sites. Mutagenesis studies confirm the critical role of the chitobiose-interacting residues in substrate binding and suggest that efficient oligosaccharide binding is required for PNGase activity. In addition, the N-terminus of a symmetry-related PNGase was found to bind to the proposed peptide-binding site of PNGase. Together with the bound chitobiose, this enables us to propose a model for glycoprotein binding to PNGase. Finally, deleting the C-terminal residues of yeast PNGase, which are disordered in all structures of this enzyme, results in a significant reduction in enzyme activity, indicating that these residues might be involved in binding of the mannose residues of the glycan chain.