PDBsum entry 2vcc

Hydrolase

PDB id: 2vcc

Contents

Protein chain

887 a.a. *

Ligands

GOL ×3

SO4

Metals

_CA

Waters ×578

* Residue conservation analysis

PDB id:

2vcc

Name:

Hydrolase

Title:

Family 89 glycoside hydrolase from clostridium perfringens

Structure:

Alpha-n-acetylglucosaminidase. Chain: a. Fragment: residues 26-916. Engineered: yes

Source:

Clostridium perfringens. Organism_taxid: 1502. Atcc: 13124. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.00Å R-factor: 0.212 R-free: 0.255

Authors:

E.Ficko-Blean,K.A.Stubbs,O.Berg,D.J.Vocadlo,A.B.Boraston

Key ref:

E.Ficko-Blean et al. (2008). Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB. Proc Natl Acad Sci U S A, 105, 6560-6565. PubMed id: 18443291 DOI: 10.1073/pnas.0711491105

Date:

19-Sep-07 Release date: 18-Mar-08

Protein chain

A0A0H2YU91  (A0A0H2YU91_CLOP1) -  Alpha-N-acetylglucosaminidase family protein from Clostridium perfringens (strain ATCC 13124 / DSM 756 / JCM 1290 / NCIMB 6125 / NCTC 8237 / Type A)

Seq: 2095 a.a.

Struc: 887 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.50 - alpha-N-acetylglucosaminidase.
• Reaction: Hydrolysis of terminal non-reducing N-acetyl-D-glucosamine residues in N-acetyl-alpha-D-glucosaminides.

DOI no: 10.1073/pnas.0711491105

Proc Natl Acad Sci U S A 105:6560-6565 (2008)

PubMed id: 18443291

Structural and mechanistic insight into the basis of mucopolysaccharidosis IIIB.

E.Ficko-Blean, K.A.Stubbs, O.Nemirovsky, D.J.Vocadlo, A.B.Boraston.

ABSTRACT

Mucopolysaccharidosis III (MPS III) has four forms (A-D) that result from buildup of an improperly degraded glycosaminoglycan in lysosomes. MPS IIIB is attributable to the decreased activity of a lysosomal alpha-N-acetylglucosaminidase (NAGLU). Here, we describe the structure, catalytic mechanism, and inhibition of CpGH89 from Clostridium perfringens, a close bacterial homolog of NAGLU. The structure enables the generation of a homology model of NAGLU, an enzyme that has resisted structural studies despite having been studied for >20 years. This model reveals which mutations giving rise to MPS IIIB map to the active site and which map to regions distant from the active site. The identification of potent inhibitors of CpGH89 and the structures of these inhibitors in complex with the enzyme suggest small-molecule candidates for use as chemical chaperones. These studies therefore illuminate the genetic basis of MPS IIIB, provide a clear biochemical rationale for the necessary sequential action of heparan-degrading enzymes, and open the door to the design and optimization of chemical chaperones for treating MPS IIIB.

Selected figure(s)

Figure 2.
Structural location of naturally occurring mutations in NAGLU. (A) A cartoon representation of the homology model of NAGLU showing its overall fold. The coloring of the domains is as for CpGH89 in Fig. 1A. (B) A structural overlay of the active site of CpGH89 and NAGLU. The catalytic residues are labeled. (C) A view of the ribbon trace of the NAGLU model with the structural location of the known mutations that lead to MPS IIIB. Sites of mutations are shown as spheres. Blue coloring indicates active site residues, whereas red coloring indicates nonactive site residues.

Figure 3.
Inhibitor binding by CpGH89. (A and B) Isotherms of CpGH89 binding to PUGNAc (A) and 2AcDNJ (B) produced by ITC. (Upper) Raw heat measurements. (Lower) Integrated heats. The solid lines show the fit of a one site-binding model to the data. (A Inset) Dixon plot analysis of CpGH89 inhibition by PUGNAc. The intersection point on the graph corresponding to the K [i] (absolute value of the X value at the intersection) is indicated by an arrow. (C and D ) Active site representations are shown for PUGNAc (C ) and 2AcDNJ (D). The blue mesh shows the maximum likelihood (43)/σ[a] (49)-weighted electron density maps contoured at 0.23 e^−/Å^3 and 0.22 e^−/Å^3 for 2AcDNJ and PUGNAc, respectively. Key active site residues, including the putative catalytic residues Glu-483 and Glu-601, are shown in stick representation and colored gray. Ligands are shown in green stick representation. Putative hydrogen bonds between the protein and ligand identified by using the criteria of proper geometry and a distance cutoff of 3.2 Å are shown as dotted magenta lines. (E and F ) Schematics showing the interactions within the active site of CpGH89 with PUGNAc (E ) and 2AcDNJ (F ). A distance of 3.2 Å was used as the cutoff for significant hydrogen bonds. Water molecules are shown as shaded spheres. Protons on the amino acids are omitted for clarity.

Figures were selected by an automated process.