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

The Pseudomonas family 10 xylanase, Xyl10A, hydrolyzes beta 1,4-linked xyla ns but exhibits very low activity against aryl-beta-cellobiosides. The fami ly 10 enzyme, Cex, from Cellulomonas fimi, hydrolyzes aryl-beta-cellobiosid es 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 ne cessary to accommodate glucose-derived substrates in the -1 subsite. We hav e evaluated the catalytic activities of the mutants Y87A, Y87F, L314A, L314 A/Y87F, and W313A of Xyl10A. Mutations to Tyr-87 increased and decreased th e catalytic efficiency against 4-nitrophenyl-beta-cellobioside and 4-nitrop henyl-beta-xylobioside, respectively The L314A mutation caused a 200-fold d ecrease in 4-nitrophenyl-beta-xylobioside activity but did not significantl y reduce 4-nitrophenyl-beta-cellobioside hydrolysis. The mutation L314A/Y87 A gave a 6500-fold improvement in the hydrolysis of glucose-derived substra tes compared with xylose-derived equivalents. These data show that substant ial improvements in the ability of Xyl10A to accommodate the C6-OH of gluco se-derived substrates are achieved when steric hindrance is removed.