Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens

Background: Unsaturated glucuronyl hydrolases (UGL) from GH88 are bacterial enzymes involved in the degradation of glycosaminoglycans and are virulence factors. Results: Mechanistic data inconsistent with the currently accepted mechanism of UGL are presented. Conclusion: A revised mechanism is proposed to explain all mechanistic data published to date. Significance: Understanding of the mechanism of UGL may allow design of bacteriostatic agents. Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct H-1 NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2.