INDUSTRIALLY ROBUST ENZYMES AND MICROORGANISMS FOR PRODUCTION OF SUGARS AND ETHANOL FROM AGRICULTURAL BIOMASS
Location: National Center for Agricultural Utilization Research
Title: Aminoalcohols as Probes of the Two-subsite Active Site of Beta-D-xylosidase from Selenomonas ruminantium
Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 18, 2008
Publication Date: December 1, 2009
Citation: Jordan, D.B., Mertens, J.A., Braker, J.D. 2009. Aminoalcohols as Probes of the Two-subsite Active Site of Beta-D-xylosidase from Selenomonas ruminantium. Biochimica et Biophysica Acta. 1794(1):144-158.
Interpretive Summary: Agricultural biomass like crop residues, grain processing byproducts, dedicated energy crops [e.g., switchgrass], etc., represent an abundant, renewable feedstock for production of ethanol and other valuable products, if practical conversion technologies can be developed. These materials are rich in complex carbohydrates that must be broken down to simple sugars before 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. 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, 10-fold more efficiently than other enzymes described by other workers. Here we report the use of aminoalcohol inhibitors to better understand the binding of sugars to the enzyme. Our results will help us and other researchers in the development of new bioconversion strategies to produce fuel ethanol economically.
Catalysis and inhibitor binding by the GH43 beta-xylosidase are governed by the protonation state of catalytic base (D14, pKa 5.0) and catalytic acid (E186, pKa 7.2) which reside in subsite -1 of the two-subsite active site. Cationic aminoalcohols are shown to bind exclusively to subsite -1 of the catalytically-inactive, dianionic enzyme (D14**-E186**-). 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). By occupying subsite -1 with ethanolamine, affinity of monosaccharides for subsite +1 is demonstrated. Inhibition patterns appear competitive, noncompetitive, and uncompetitive depending on the strength of enzyme-aminoalcohol-substrate complexes. Biphasic inhibition by triethanolamine reveals minor (<0.01%) contaminations of E186A preparations (including a (His)6-Tagged form) by wild-type-like enzyme, likely originating from translational misreading. Aminoalcohols can be useful in probing glycoside hydrolases; they can cause artifacts when used unwarily.