Crystal structures of Geobacillus stearothermophilus α-glucuronidase complexed with its substrate and products: Mechanistic implications

α-Glucuronidases cleave the α-1,2-glycosidic bond between 4-O-methyl-D-glucuronic acid and short xylooligomers as part of the hemicellulose degradation system. To date, all of the α-glucuronidases are classified as family 67 glycosidases, which catalyze the hydrolysis via the investing mechanism. Here we describe several high resolution crystal structures of the α-glucuronidase (AguA) from Geobacillus stearothermophilus, in complex with its substrate and products. In the complex of AguA with the intact substrate, the 4-O-methyl-D-glucuronic acid sugar ring is distorted into a half-chair conformation, which is closer to the planar conformation required for the oxocarbenium ion-like transition state structure. In the active site, a water molecule is coordinated between two carboxylic acids, in an appropriate position to act as a nucleophile. From the structural data it is likely that two carboxylic acids, Asp³⁶⁴ and Glu ³⁹², activate together the nucleophilic water molecule. The loop carrying the catalytic general acid Glu²⁸⁵ cannot be resolved in some of the structures but could be visualized in its "open" and "closed" (catalytic) conformations in other structures. The protonated state of Glu²⁸⁵ is presumably stabilized by its proximity to the negative charge of the substrate, representing a new variation of substrate-assisted catalysis mechanism.