PDBsum entry 1w3l

Hydrolase

PDB id: 1w3l

Contents

Protein chain

300 a.a. *

Ligands

BGC-BGC

GOL ×2

SO4

OXZ

Waters ×606

* Residue conservation analysis

PDB id:

1w3l

Name:

Hydrolase

Title:

Endoglucanase cel5a from bacillus agaradhaerens in complex with cellotri derived-tetrahydrooxazine

Structure:

Endoglucanase 5a. Chain: a. Fragment: catalytic module, residues 27-329. Synonym: endo-1,4-beta-glucanase, alkaline cellulase. Engineered: yes

Source:

Bacillus agaradhaerens. Organism_taxid: 76935. Expressed in: bacillus subtilis. Expression_system_taxid: 1423.

Resolution:

1.04Å R-factor: 0.106 R-free: 0.121

Authors:

T.M.Gloster,J.M.Macdonald,C.A.Tarling,R.V.Stick,S.W.Withers, G.J.Davies

Key ref:

T.M.Gloster et al. (2004). Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases. J Biol Chem, 279, 49236-49242. PubMed id: 15356002 DOI: 10.1074/jbc.M407195200

Date:

16-Jul-04 Release date: 08-Sep-04

Protein chain

O85465  (GUN5_SALAG) -  Endoglucanase 5A from Salipaludibacillus agaradhaerens

Seq: 400 a.a.

Struc: 300 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.4 - cellulase.
• Reaction: Endohydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, lichenin and cereal beta-D-glucans.

DOI no: 10.1074/jbc.M407195200

J Biol Chem 279:49236-49242 (2004)

PubMed id: 15356002

Structural, thermodynamic, and kinetic analyses of tetrahydrooxazine-derived inhibitors bound to beta-glucosidases.

T.M.Gloster, J.M.Macdonald, C.A.Tarling, R.V.Stick, S.G.Withers, G.J.Davies.

ABSTRACT

The understanding of transition state mimicry in glycoside hydrolysis is increasingly important both in the quest for novel specific therapeutic agents and for the deduction of enzyme function and mechanism. To aid comprehension, inhibitors can be characterized through kinetic, thermodynamic, and structural dissection to build an "inhibition profile." Here we dissect the binding of a tetrahydrooxazine inhibitor and its derivatives, which display Ki values around 500 nm. X-ray structures with both a beta-glucosidase, at 2 A resolution, and an endoglucanase at atomic (approximately 1 A) resolution reveal similar interactions between the tetrahydrooxazine inhibitor and both enzymes. Kinetic analyses reveal the pH dependence of kcat/Km and 1/Ki with both enzyme systems, and isothermal titration calorimetry unveils the enthalpic and entropic contributions to beta-glucosidase inhibition. The pH dependence of enzyme activity mirrored that of 1/Ki in both enzymes, unlike the cases of isofagomine and 1-deoxynojirimycin that have been characterized previously. Calorimetric dissection reveals a large favorable enthalpy that is partially offset by an unfavorable entropy upon binding. In terms of the similar profile for the pH dependence of 1/Ki and the pH dependence of kcat/Km, the significant enthalpy of binding when compared with other glycosidase inhibitors, and the tight binding at the optimal pH of the enzymes tested, tetrahydrooxazine and its derivatives are a significantly better class of glycosidase inhibitor than previously assumed.

Selected figure(s)

Figure 2.
FIG. 2. Aza sugar glycosidase inhibitors.

Figure 4.
FIG. 4. Divergent (wall-eyed) stereo ball-and-stick representation of TmGH1 active site residues interacting with 1 (a) and Cel5 active site residues interacting with 2 (b). The observed electron density for the maximum likelihood weighted 2F[obs] - F[calc] map is contoured at 1 ( 0.25 electrons Å-3) for TmGH1 and 2.5 ( 1.16 electrons Å-3) for Cel5A; these figures were drawn using BOBSCRIPT (31).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 49236-49242) copyright 2004.

Figures were selected by an automated process.