B-d-Xylosidase from Selenomonas ruminantium: Catalyzed Reactions with Natural and Artificial Substrates

Abstract

Catalytically efficient β-d-xylosidase from Selenomonas ruminantium (SXA) exhibits pK as 5 and 7 (assigned to catalytic base, D14, and catalytic acid, E186) for k cat/K m with substrates 1,4-β-d-xylobiose (X2) and 1,4-β-d-xylotriose (X3). Catalytically inactive, dianionic SXA (D14−E186−) has threefold lower affinity than catalytically active, monoanionic SXA (D14−E186H) for X2 and X3, whereas D14−E186− has twofold higher affinity than D14−E186H for 4-nitrophenyl-β-d-xylopyranoside (4NPX), and D14−E186− has no affinity for 4-nitrophenyl-α-l-arabinofuranoside. Anomeric isomers, α-d-xylose and β-d-xylose, have similar affinity for SXA. 4-Nitrophenol competitively inhibits SXA-catalyzed hydrolysis of 4NPX. SXA steady-state kinetic parameters account for complete progress curves of SXA-catalyzed hydrolysis reactions.KeywordsFuel ethanolGlycoside hydrolaseGH43HemicellulosepH dependenceStereochemistryInhibitorAssay method