PDBsum entry: 1xw2

Hydrolase

PDB id: 1xw2

Contents

Protein chain

404 a.a. *

Waters ×514

* Residue conservation analysis

PDB id:

1xw2

Name:

Hydrolase

Title:

Structure of a cold-adapted family 8 xylanase

Structure:

Endo-1,4-beta-xylanase. Chain: a. Engineered: yes. Mutation: yes

Source:

Pseudoalteromonas haloplanktis. Organism_taxid: 228. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

1.76Å R-factor: 0.161 R-free: 0.178

Authors:

T.Collins,D.De Vos,A.Hoyoux,S.N.Savvides,C.Gerday,J.Van Beeumen,G.Feller

Key ref:

T.Collins et al. (2005). Study of the active site residues of a glycoside hydrolase family 8 xylanase. J Mol Biol, 354, 425-435. PubMed id: 16246370 DOI: 10.1016/j.jmb.2005.09.064

Date:

29-Oct-04 Release date: 11-Oct-05

Protein chain

Q8RJN8  (Q8RJN8_PSEHA) -  Endo-1,4-beta-xylanase (Precursor)

Seq: 426 a.a.

Struc: 404 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.2.1.8 - Endo-1,4-beta-xylanase.
• Reaction: Endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans.

 Gene Ontology (GO) functional annotation 

• Biological process: metabolic process (3 terms)
• Biochemical function: catalytic activity (5 terms)

DOI no: 10.1016/j.jmb.2005.09.064

J Mol Biol 354:425-435 (2005)

PubMed id: 16246370

Study of the active site residues of a glycoside hydrolase family 8 xylanase.

T.Collins, D.De Vos, A.Hoyoux, S.N.Savvides, C.Gerday, J.Van Beeumen, G.Feller.

ABSTRACT

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.

Selected figure(s)

Figure 4.
Figure 4. Superposition of the principal active site residues (sticks) and the proposed nucleophilic water (spheres) of wild-type pXyl (grey) with those of mutants (color-coded according to atom type): (a) E78Q; (b) D281N; (c) D144N; and (d) D144A. The hydrogen bonds and nucleophilic water in the mutants are indicated by broken lines and a red sphere, respectively, while the nucleophilic water in the wild-type pXyl is indicated by a grey sphere.

Figure 5.
Figure 5. Structural comparison of endoglucanase CelA and endoxylanase pXyl. (a) Detailed view of the catalytic centre of CelA mutant E95Q with glucose residues at subsites -1 and +1 and the most important catalytic residues superimposed on the equivalent residues of WT pXyl. Carbon atoms of CelA and pXyl are coloured yellow and grey, respectively. The proposed nucleophilic water molecules of CelA and pXyl are indicated by spheres. The distance between the nucleophilic water of CelA and the anomeric carbon C1 at subsite -1 is indicated by a dotted line. Hydrogen bonds are indicated by broken lines and distances are in Å. (b) Stereo-drawing of the catalytic site of CelA (E95Q) in complex with cellopentaose (subsites -3 to +2) and cellotriose (subsites +1 to +3), and superposition on the equivalent residues of WT pXyl. Oxygen atoms are coloured red, nitrogen atoms are coloured blue and carbon atoms are coloured yellow (CelA) or green (pXyl).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2005, 354, 425-435) copyright 2005.

Figures were selected by the author.