Journal
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS
Volume 1814, Issue 12, Pages 1648-1657Publisher
ELSEVIER
DOI: 10.1016/j.bbapap.2011.08.010
Keywords
Site-directed mutagenesis; Pyranosyl substrate distortion; Glycoside hydrolase; Plant cell wall deconstruction; Biomass saccharification
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Conformational inversion occurs 7-8 kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA)>4-nitrophenyl-beta-D-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite -1 residues decrease the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support other factors, such as Cl migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise k(cat)(X2/4NPX) and alanine mutations of subsite +1 and interface residues lower k(cat)(X2/4NPX). We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite -1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues. (C) 2011 Elsevier B.V. All rights reserved.
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