Submitted to: Archives Of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2008
Publication Date: 4/1/2008
Citation: Brunzelle, J.S., Jordan, D.B., McCaslin, D.R., Olczak, A., Wawrzak, Z. 2008. Structure of the two-subsite beta-D-xylosidase from Selenomonas ruminantium in complex with 1,3-bis[tris(hydroxymethyl)methylamino] propane. Archives Of Biochemistry and Biophysics. 474(1):157-166. 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 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, 10-fold more efficiently than other enzymes described by other workers. Here, we report the three-dimensional structure of the enzyme in complex with an active-site inhibitor. Also, we report on hydrodynamic studies that determined a dissociation constant for the tetrameric enzyme to the dimeric enzyme. Our results will help us and other researchers in the development of new bioconversion strategies to produce fuel ethanol economically.
Technical Abstract: The three-dimensional structure of the catalytically-efficient beta-xylosidase from Selenomonas ruminantium in complex with competitive inhibitor 1,3 bis[tris(hydroxymethyl)methylamino]propane (BTP) was determined by using X-ray crystallography (1.3 Å resolution). Most H bonds between inhibitor and protein occur within subsite -1, including one between the carboxyl group of E186 and an N group of BTP. The other N of BTP occupies subsite +1 near K99. D14 (pKa 5.0) and E186 (pKa 7.2) serve as catalytic base and catalytic acid, respectively. The pH (6-10) profile for 1/Ki**(BTP) is bell-shaped with pKa’s 7.0 and 7.8 on the acidic limb and 9.8 on the basic limb. Mutation K99A eliminates pKa 7.8, strongly suggesting that the BTP monocation binds to the dianionic enzyme D14**-E186**-. A sedimentation equilibrium experiment estimates a Kd ([dimer]**2/[tetramer]) of 7 x 10**-9 M. Similar kcat and kcat/Km values were determined when the tetramer/dimer ratio changes from 0.0028 to 26.