Toggle Main Menu Toggle Search

Open Access padlockePrints

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

Lookup NU author(s): Simon Andrews, Professor Jeremy LakeyORCiD, Emeritus Professor Harry Gilbert


Full text for this publication is not currently held within this repository. Alternative links are provided below where available.


The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes β1,4-linked xylans but exhibits very low activity against aryl-β-cellobiosides. The family 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-β-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-β-cellobioside and 4-nitrophenyl-β-xylobioside, respectively. The L314A mutation caused a 200-fold decrease in 4-nitrophenyl-β-xylobioside activity but did not significantly reduce 4-nitrophenyl-β-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.

Publication metadata

Author(s): Andrews SR; Lakey JH; Gilbert HJ; Charnock SJ; Davies GJ; Claeyssens M; Nerinckx W; Underwood M; Sinnott ML; Warren RAJ

Publication type: Article

Publication status: Published

Journal: Journal of Biological Chemistry

Year: 2000

Volume: 275

Issue: 30

Pages: 23027-23033

ISSN (print): 0021-9258

ISSN (electronic): 1083-351X

Publisher: American Society for Biochemistry and Molecular Biology


DOI: 10.1074/jbc.M000128200

PubMed id: 10767281


Altmetrics provided by Altmetric