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 J Warren, H J Gilbert

Research output: Contribution to journalArticlepeer-review

Abstract

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes beta 1,4-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.

Original languageEnglish
Pages (from-to)23027-23033
Number of pages7
JournalJournal of Biological Chemistry
Volume275
Issue number30
Publication statusPublished - 28 Jul 2000

Keywords

  • FLUORESCENS SUBSPECIES CELLULOSA
  • BETA-1,4-GLYCANASE CEX
  • DIRECTED MUTAGENESIS
  • CRYSTAL-STRUCTURE
  • SEQUENCE
  • ENZYME
  • FIMI
  • INTERMEDIATE
  • EXPRESSION
  • PROTEIN

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