PDBsum entry: 1e0w

Glycoside hydrolase family 10

PDB id: 1e0w

Contents

Protein chain

302 a.a. *

Waters ×438

* Residue conservation analysis

PDB id:

1e0w

Name:

Glycoside hydrolase family 10

Title:

Xylanase 10a from sreptomyces lividans. Native structure at 1.2 angstrom resolution

Structure:

Xylanase a. Chain: a. Fragment: catalytic module. Engineered: yes

Source:

Streptomyces lividans. Organism_taxid: 1916. Expressed in: streptomyces lividans. Expression_system_taxid: 1916.

Resolution:

1.2Å R-factor: 0.098 R-free: 0.124

Authors:

V.Ducros,S.J.Charnock,U.Derewenda,Z.S.Derewenda,Z.Dauter, C.Dupont,F.Shareck,R.Morosoli,D.Kluepfel,G.J.Davies

Key ref:

S.R.Andrews et al. (2000). Substrate specificity in glycoside hydrolase family 10. Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of Pseudomonas cellulosa xylanase 10A. J Biol Chem, 275, 23027-23033. PubMed id: 10767281 DOI: 10.1074/jbc.M000128200

Date:

10-Apr-00 Release date: 05-Apr-01

Protein chain

P26514  (XYNA_STRLI) -  Endo-1,4-beta-xylanase A

Seq: 477 a.a.

Struc: 302 a.a.

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: carbohydrate metabolic process (1 term)
• Biochemical function: catalytic activity (3 terms)

DOI no: 10.1074/jbc.M000128200

J Biol Chem 275:23027-23033 (2000)

PubMed id: 10767281

Substrate specificity in glycoside hydrolase family 10. Tyrosine 87 and leucine 314 play a pivotal role in discriminating between glucose and xylose binding in the proximal active site of Pseudomonas cellulosa xylanase 10A.

S.R.Andrews, S.J.Charnock, J.H.Lakey, G.J.Davies, M.Claeyssens, W.Nerinckx, M.Underwood, M.L.Sinnott, R.A.Warren, H.J.Gilbert.

ABSTRACT

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes beta1, 4-linked xylans but exhibits very low activity against aryl-beta-cellobiosides. The family 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-beta-cellobiosides more efficiently than does Xyl10A, and the movements of two residues in the -1 and -2 subsites are implicated in this relaxed substrate specificity (Notenboom, V., Birsan, C., Warren, R. A. J., Withers, S. G., and Rose, D. R. (1998) Biochemistry 37, 4751-4758). The three-dimensional structure of Xyl10A suggests that Tyr-87 reduces the affinity of the enzyme for glucose-derived substrates by steric hindrance with the C6-OH in the -2 subsite of the enzyme. Furthermore, Leu-314 impedes the movement of Trp-313 that is necessary to accommodate glucose-derived substrates in the -1 subsite. We have evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased the catalytic efficiency against 4-nitrophenyl-beta-cellobioside and 4-nitrophenyl-beta-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-beta-xylobioside activity but did not significantly reduce 4-nitrophenyl-beta-cellobioside hydrolysis. The mutation L314A/Y87A gave a 6500-fold improvement in the hydrolysis of glucose-derived substrates compared with xylose-derived equivalents. These data show that substantial improvements in the ability of Xyl10A to accommodate the C6-OH of glucose-derived substrates are achieved when steric hindrance is removed.

Selected figure(s)

Figure 2.
Fig. 2. Divergent (wall-eyed) stereo representation of the -2 and -1 subsites of Cex, Xyl10A , and SlXyl10A. Shown are subsites of the C. fimi enzyme Cex (cellobiosyl enzyme in blue, xylobiosyl enzyme in pale green) (14, 15) and the P. cellulosa Xyl10A (red). Residues mutated in this study are labeled. This figure was prepared with QUANTA (Molecular Simulations Inc., San Diego, CA).

Figure 4.
Fig. 4. CD spectroscopy of native and mutant forms of Xyl10A. Native Xyl10A (green) and the mutants L314A (red), Y87F (blue), L314A/Y87F (purple), and W313A (black) were subjected to CD spectroscopy as described previously (26).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2000, 275, 23027-23033) copyright 2000.

Figures were selected by an automated process.