Sunday, April 29, 2007
2-25

ß-D-Xylosidase from Selenomonas ruminantium: thermodynamics of enzyme-catalyzed and noncatalyzed reactions

Douglas Jordan, Fermentation Biotechnology, USDA-ARS, 1815 N. University Street, Peoria, IL 61604

β-D-Xylosidase from Selenomonas ruminantium has been revealed as the best catalyst known for promoting hydrolysis of 1,4-β-D-xylooligosaccharides and it has potential utility in saccharification processes. Values of kcat and kcat/Km are more than 10-fold larger than those reported for the enzyme from other organisms. The family 43 glycoside hydrolase acts through a single transition state, inversion mechanism, and cleaves a single xylose residue from the nonreducing end of xylooligosaccharides per catalytic cycle without processivity. Three-dimensional structures of homologous GH43 xylosidases indicate that the enzyme active site has only two subsites for recognition of substrate, the two terminal xylosyl residues that share the scissile glycosidic bond.  In addition to its xylosidase activity, the enzyme efficiently catalyzes hydrolysis of 4-nitrophenyl-α-L-arabinofuranoside. Individual mutations of the catalytic base (ASP14) and catalytic acid (Glu186) to alanine erode kcat and kcat/Km by > 103 and to a similar extent for substrates 4-nitrophenyl-β-D-xylopyranoside and 4-nitrophenyl-α-L-arabinofuranoside, indicating that the two substrates share the same active site. Temperature dependence of kinetic parameters of enzyme-catalyzed reactions and noncatalyzed reactions were determined.

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