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ARS Home » Research » Publications at this Location » Publication #191754
Title: STRUCTURE-FUNCTION RELATIONSHIPS OF A CATALYTICALLY EFFICIENT BETA-D-XYLOSIDASE

Author
item Jordan, Douglas
item Li, Xin Liang
item Dunlap, Christopher
item Whitehead, Terence
item Cotta, Michael

Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/11/2006
Publication Date: 3/1/2007
Citation: Jordan, D.B., Li, X.-L., Dunlap, C.A., Whitehead, T.R., Cotta, M.A. 2007. Structure-function relationships of a catalytically efficient beta-D-xylosidase. Applied Biochemistry and Biotechnology. 141:51-76.

Interpretive Summary: Agricultural biomass like 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 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 similar enzymes described by other workers. Our results will help us and other researchers in the development of new bioconversion strategies to produce fuel ethanol economically.

Technical Abstract: Beta-D-xylosidase from the ruminal anaerobic bacterium, Selenomonas ruminantium, is revealed as the best catalyst known (kcat, kcat/Km) for promoting hydrolysis of 1,4-beta-D-xylooligosaccharides. 1H NMR experiments indicate that the family 43 glycoside hydrolase acts through an inversion mechanism on substrates 4-nitrophenyl-beta-D-xylopyranoside and 1,4-beta-D-xylobiose (X2). Progress curves of 4-nitrophenyl-beta-D-xylobioside, xylotetraose, and xylohexaose reactions, monitoring temporal accumulation of hydrolysis products, strongly suggest the enzyme-catalyzed reactions proceed by cleaving one residue from the nonreducing end of substrate per catalytic cycle without processivity. Values of kcat and kcat/Km decrease for xylooligosaccharides longer than X2, illustrating the importance to catalysis of the -1 and +1 subsites and the lack there of the +2 subsite. 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 defined; they suggest that D14 and E186 serve catalysis as general base and general acid, respectively. Individual mutation of D14 and E186 to alanine degrades kcat by > 10**3, confirming their significance. pH governs catalysis with pKa values of 5 and 7, assigned to D14 and E186, respectively. D14**HE187**H enzyme neither binds substrates nor catalyzes, D14**–E187**H binds substrates and inhibitors and catalyzes, and D14**–E187**– cannot catalyze but it binds competitive inhibitors and substrate 4-nitrophenyl-beta-D-xylopyranoside (but not substrate 4-nitrophenyl-alpha-L-arabinofuranoside). In contrast to other competitive inhibitors examined, L-arabinose exhibits slow binding kinetics and can bind two molecules per active site of D14**–E187**H enzyme; L-arabinose binds rapidly and one molecule per active site of D14**–E187**– enzyme.