Location: Bioenergy Research Unit
Title: Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase Authors
Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 18, 2011
Publication Date: November 1, 2011
Citation: Jordan, D.B., Braker, J.D. 2011. Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase. Biochimica et Biophysica Acta. 1814:1648-1657. Interpretive Summary: Agricultural biomass including crop residues, grain processing byproducts, dedicated energy crops (e.g. switchgrass), etc., represent abundant, renewable feedstocks for production of ethanol and other valuable products if practical conversion technologies can be developed. These materials are rich in complex carbohydrates that must first be broken down to simple sugars that can be fermented by microorganisms to ethanol and other products. A critical step in the development of new conversion processes is the discovery and development of cost efficient enzymes to convert these complex materials to simple sugars. We have discovered an enzyme involved in the final step in the hydrolysis of xylan, the second most abundant carbohydrate in plants. This enzyme produces the simple sugar, D-xylose, more efficiently than counterpart enzymes described by others. The enzyme also catalyzes the release of L-arabinose from arabinosides. This work describes detailed experiments on single-site directed mutants of all the enzyme active-site residues to better understand how the enzyme works on xylooligosaccharides. Our results will help the development of new bioconversion strategies to produce fuel ethanol economically.
Technical Abstract: Conformational inversion occurs 7-8 kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to disclose active-site residues involved in distorting substrate towards 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 elements of the glycone and aglycone 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 glycone and aglycone moieties of substrate demonstrates that the mutation of subsite -1 residues decreases the ratio kcat**4NPX/4NPA, suggesting that the native residues support substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio kcat**4NPX/4NPA, suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise kcat**X2/4NPX and alanine mutations of subsite +1 and interface residues lower kcat**X2/4NPX. Thus, substrate distortion is supported mainly by native residues of subsite -1. Other factors leading to the transition state are supported mainly by native residues of subsite +1 and interface residues.