Submitted to: Applied Biochemistry and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 25, 2009
Publication Date: February 3, 2010
Citation: Jordan, D.B., Braker, J.D. 2010. Beta-D-xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-directed Mutagenesis, Alternate Substrates, and Active-site Inhibitor. Applied Biochemistry and Biotechnology. 161(1-8):395-410. Interpretive Summary: Agricultural biomass such as crop residues, grain processing byproducts, and dedicated energy crops, represent abundant, renewable feedstock for production of ethanol and other bioproducts if practical conversion technologies can be developed. These materials are rich in complex carbohydrates that must first be broken down to simple sugars for subsequent fermentation 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 in efficient and economical manner. We have discovered an enzyme responsible for catalyzing the final step in the hydrolysis of xylan, the second most abundant carbohydrate in certain plants. This enzyme produces the simple sugar, xylose, more efficiently than counterpart enzymes described by other workers. The enzyme also catalyzes the release of arabinose from arabinosides. This work elaborates on the role of the enzyme residue, glutamate 186, in serving as a general acid in catalyzing hydrolysis reactions. Our results will help the development of new bioconversion strategies to produce fuel ethanol economically.
Technical Abstract: Beta-D-xylosidase/alpha-L-arabinofuranosidase from Selenomonas ruminantium (SXA) is the most active enzyme known for catalyzing hydrolysis of 1,4-beta-D-xylooligosaccharides to D xylose. Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation states of catalytic base (D14, pKa 5.0) and catalytic acid (E186, pKa 7.2). Biphasic inhibition by triethanolamine reveals minor (<0.03%) contamination of E186A preparations by wild-type-like enzyme, the contaminant likely originating from translational misreading. Titration of E186A preparations with triethanolamine allows resolution of binding and kinetic parameters of the E186A mutant from those of the contaminant. The E186A mutation abolishes the pKa assigned to E186; mutant enzyme binds only the neutral aminoalcohol (pH-independent Ki**triethanolamine=19 mM), whereas wild-type enzyme binds only the cationic aminoalcohol (pH-independent Ki**triethanolamine=0.065 mM). At pH 7.0 and 25 deg C, relative kinetic parameters (kcat**4NPX/kcat**4NPA and kcat/Km**4NPX/kcat/Km**4NPA) for substrates 4-nitrophenyl-beta-D-xylopyranoside (4NPX) and 4-nitrophenyl-alpha-L-arabinofuranoside (4NPA) of the E186A SXA are, respectively, 30-fold and 2-fold that of wild-type enzyme, consistent with the view that, on the enzyme, protonation is of greater importance to the transition state of 4NPA whereas ring deformation dominates the transition state of 4NPX.