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 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.
Andrews
S R
SR
Department of Biological and Nutritional Sciences, University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, United Kingdom.
Charnock
S J
SJ
Lakey
J H
JH
Davies
G J
GJ
Claeyssens
M
M
Nerinckx
W
W
Underwood
M
M
Sinnott
M L
ML
Warren
R A
RA
Gilbert
H J
HJ
D001483:Base Sequence; D002475:Cellobiose / Q000031:analogs & derivatives / Q000378:metabolism; D002942:Circular Dichroism; D017931:DNA Primers; D005947:Glucose / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007930:Leucine / Q000378:metabolism*; D011549:Pseudomonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014443:Tyrosine / Q000378:metabolism*; D043325:Xylan Endo-1,3-beta-Xylosidase; D014990:Xylans / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*; D014995:Xylosidases / Q000737:chemistry / Q000378:metabolism*
D017931:DNA Primers; D011994:Recombinant Proteins; D014990:Xylans; C074099:cellobiosyl fluoride; D002475:Cellobiose; D014443:Tyrosine; D014994:Xylose; D006026:Glycoside Hydrolases; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D007930:Leucine; D005947:Glucose