PDBsum entry: 2osy

Hydrolase

PDB-id: 2osy

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Description

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PROCHECK

Protein chains

432 a.a. *

Ligands

GLC-GAL ×2

Metal ions

_NA ×2

Waters ×301

* Residue conservation analysis

Tools

Clefts Calculation

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PDB id:

2osy

Name:

Hydrolase

Title:

Endo-glycoceramidase ii from rhodococcus sp.: Lactosyl- enzyme intermediate

Structure:

Endoglycoceramidase ii. Chain: a, b. Engineered: yes. Mutation: yes

Source:

Rhodococcus sp.. Organism_taxid: 1831. Strain: m-777. Expressed in: escherichia coli. Expression_system_taxid: 562.

UniProt:

Chains A, B: O33853 (O33853_RHOSO)

Seq:

Struc:

Seq:

490 a.a.

Struc:

432 a.a.*

Key:

PfamA domain

Secondary structure

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

Enzyme class:

E.C.3.2.1.123   [IntEnz]   [ExPASy]   [KEGG]   [BRENDA]

Reaction:

Oligoglycosylglucosylceramide + H₂O = ceramide + oligoglycosylglucose (see diagram below)

Resolution:

2.10Å

R-factor:

0.208

R-free:

0.251

Authors:

M.E.C.Caines,N.C.J.Strynadka

Key ref:

M.E.Caines et al. (2007). Structural and mechanistic analyses of endo-glycoceramidase II, a membrane-associated family 5 glycosidase in the Apo and GM3 ganglioside-bound forms.. J Biol Chem, 282, 14300-14308. [PubMed id: 17329247] [DOI: 10.1074/jbc.M611455200]

Date:

06-Feb-07

Release date:

27-Feb-07

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Clefts

Surface

Key reference

DOI no: 10.1074/jbc.M611455200

J Biol Chem 282:14300-14308 (2007)

PubMed id: 17329247

Structural and mechanistic analyses of endo-glycoceramidase II, a membrane-associated family 5 glycosidase in the Apo and GM3 ganglioside-bound forms.

M.E.Caines, M.D.Vaughan, C.A.Tarling, S.M.Hancock, R.A.Warren, S.G.Withers, N.C.Strynadka.

ABSTRACT

endo-Glycoceramidase, a membrane-associated family 5 glycosidase, deviates from the typical polysaccharide substrate specificity of other soluble members of the family, preferentially hydrolyzing glycosidic linkages between the oligosaccharide and ceramide moieties of gangliosides. Here we report the first x-ray crystal structures of an endo-glycoceramidase from Rhodococcus sp., in the apo form, in complex with the ganglioside G(M3) (Svennerholm ganglioside nomenclature (Svennerholm, L. (1964) J. Lipid Res. 5, 145-155)), and trapped as a glycosyl-enzyme intermediate. These snapshots provide the first molecular insight into enzyme recognition and association with gangliosides, revealing the structural adaptations necessary for glycosidase-catalyzed hydrolysis and detailing a novel ganglioside binding topology. Consistent with the chemical duality of the substrate, the active site of endo-glycoceramidase is split into a wide, polar cavity to bind the polyhydroxylated oligosaccharide moiety and a narrow, hydrophobic tunnel to bind the ceramide lipid chains. The specific interactions with the ceramide polar head group manifest a surprising aglycone specificity, an observation substantiated by our kinetic analyses. Collectively, the reported structural and kinetic data provide insight toward rational redesign of the synthetic glycosynthase mutant of endo-glycoceramidase to enable facile synthesis of nonnatural, therapeutically useful gangliosides.

Selected figure(s)

Figure 2.
FIGURE 2. a, the structure of the EGC monomer. b, the electrostatic surface potential of EGC (red, electronegative; blue, electropositive; contoured from -15 to 1 kT/e). c, the hydrophobic surface potential of EGC (green, hydrophobic; white, polar). d, the structure of the -(1,4)-glucanase from Bacillus agaradherans, Cel5A (Protein Data Bank code 2A3H). e, the electrostatic surface potential of Cel5A (red, electronegative; blue, electropositive; contoured from -20 to 1 kT/e). Bound ligands, G[M3] (a and b) and cellobiose (d and e), are shown as ball-and-stick representations in yellow.

Figure 3.
FIGURE 3. a, electron density for the bound G[M3].AnmF[o] - DF[c] (36) electron density map, calculated after random model perturbation and refinement with G[M3] atoms omitted, is shown contoured around the G[M3] at 2.5 in red. b, a surface representation depicting the G[M3] binding site. G[M3] is shown as a ball-and-stick representation in yellow, surrounded by its ligands in the active site of EGC. c, a schematic representation of polar, close contacts involved in the binding of G[M3]. Water molecules are represented by gray spheres.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 14300-14308) copyright 2007.

Figures were selected by an automated process.