Catalytically Efficient β-D-Xylosidase 51 Applied Biochemistry and Biotechnology Vol. 141 2007 Copyright © 2007 by Humana Press Inc. All rights of any nature whatsoever reserved. 0273-2289/1559-0291 (Online)/07/141:51–76/$30.00 51 *Author to whom all correspondence and reprint requests should be addressed. Structure–Function Relationships of a Catalytically Efficient β-D-Xylosidase DOUGLAS B. JORDAN,* ,1 XIN-LIANG LI, 1 CHRISTOPHER A. DUNLAP, 2 TERENCE R. WHITEHEAD, 1 AND MICHAEL A. COTTA 1 1 Fermentation Biotechnology Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Agricultural Research Service, † 1815 N. University Street, Peoria, IL 61604, E-mail: jordand@ncaur.usda.gov; 2 Crop Bioprotection Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604 Received May 22, 2006; Revised July 6, 2006; Accepted July 11, 2006 Abstract β-D-Xylosidase from Selenomonas ruminantium is revealed as the best catalyst known (k cat , k cat /K m ) for promoting hydrolysis of 1,4-β-D-xylooligosaccharides. 1 H nuclear magnetic resonance experiments indicate the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl- β-D-xylopyranoside (4NPX) and 1,4-β-D-xylobiose (X2). Progress curves of 4-nitrophenyl-β-D-xylobioside, xylotetraose and xylohexaose reactions indicate that one residue from the nonreducing end of substrate is cleaved per catalytic cycle without processivity. Values of k cat and k cat /K m decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of subsites –1 and +1 and the lack there of subsite +2. Homology models of the enzyme active site with docked substrates show that subsites beyond –1 are blocked by protein and subsites beyond +1 are not formed; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutations, D14A and E186A, erode k cat and k cat /K m by <10 3 and to a similar extent for substrates 4NPX and 4-nitrophenyl-α-L-arabinofuranoside † The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.