|Li, Xin liang|
Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer reviewed journal
Publication Acceptance Date: 6/25/2007
Publication Date: 7/6/2007
Citation: Jordan, D.B., Li, X. 2007. Variation in relative substrate specificity of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition. Biochimica et Biophysica Acta. 1774(9):1192-1198. Interpretive Summary: Agricultural biomass like crop residues, grain processing byproducts, dedicated energy crops (e.g., switchgrass), etc., represent an abundant, renewable feedstock 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 brokendown 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 new 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, xylose, more efficiently than other enzymes described by other workers. The enzyme also catalyzes the release of arabinose from arabinosides. This work reveals enzyme residues that control the ratio of xylosidase and arabinosidase activities. The residues are involved in substrate recognition and in substrate distortion so that the reactions occur. Our results will help us and other researchers in the development of new bioconversion strategies to produce fuel ethanol economically.
Technical Abstract: 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 (subsite -1) of the bifunctional 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 by factors of 330 to 280,000, 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 respective losses in (kcat/Km)**4NPX of 163,000, 287, and 48.3 fold and with respective losses in (kcat/Km)**4NPA of 1,760, 9.48, and 17.7 fold. In active-site models, 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.