Variation in relative substrate specificity of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition.
To probe differential control of substrate specificities for 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA) and 4-nitrophenyl-beta-d-xylopyranoside (4NPX), residues of the glycone binding pocket of GH43 beta-d-xylosidase/alpha-l-arabinofuranosidase from Selenomonas ruminantium were individually mutated to alanine. Although their individual substrate specificities (kcat/Km)(4NPX) and (kcat/Km)(4NPA) are lowered 330 to 280,000 fold, D14A, D127A, W73A, E186A, and H248A mutations maintain similar relative substrate specificities as wild-type enzyme. Relative substrate specificities (kcat/Km)(4NPX)/(kcat/Km)(4NPA) are lowered by R290A, F31A, and F508A mutations to 0.134, 0.407, and 4.51, respectively, from the wild type value of 12.3 with losses in (kcat/Km)(4NPX) and (kcat/Km)(4NPA) of 18 to 163000 fold. R290 and F31 reside above and below the C4 OH group of 4NPX and the C5 OH group of 4NPA, where they can serve as anchors for the two glycone moieties when their ring systems are distorted to transition-state geometries by raising the position of C1. Thus, whereas R290 and F31 provide catalytic power for hydrolysis of both substrates, the native residues are more important for 4NPX than 4NPA as the xylopyranose ring must undergo greater distortion than the arabinofuranose ring. F508 borders C4 and C5 of the two glycone moieties and can serve as a hydrophobic platform having more favorable interactions with xylose than arabinofuranose.
Jordan
Douglas B
DB
Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA. douglas.jordan@ars.usda.gov
Li
Xin-Liang
XL
D001665:Binding Sites; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D013379:Substrate Specificity; D014995:Xylosidases / Q000378:metabolism*
D006026:Glycoside Hydrolases; D014995:Xylosidases; C019713:alpha-N-arabinofuranosidase