Insights into the Catalytic Mechanism of Unsaturated Glucuronyl Hydrolase of Bacillus sp. GL1.

Together with polysaccharide lyases (PLs), the unsaturated glucuronyl hydrolase of Bacillus sp., GL1, is responsible for the metabolism of glycosaminoglycans (GAGs), which plays an important role in various crucial physiological events. More importantly, the degradation mechanism of GAGs often causes extracellular bacterial infection and is thought to be one of the virulence factors. We have previously studied the first degradation step catalyzed by PLs. In this work, we focused on the degradation of the unsaturated chondroitin disaccharide, which is produced from chondroitin by chondroitin lyase. A combined quantum mechanical and molecular mechanical method was employed in all simulations. First of all, molecular dynamics simulations were performed to obtain a stable initial enzyme-substrate complex structure. Almost all interactions between the substrate and enzyme were found to be related to the d-glucuronic acid unit, whereas no recognition specificity was observed for the N-acetyl-d-galactosamine unit. Experimentally, two different pathways have been proposed on the basis of X-ray structures and kinetic isotopic effects. In our simulation, the pathway involving the formation of an epoxide intermediate has been found to be favorable rather than that involving direct hydration of the vinyl ether group around carbons 4 and 5. A metastable oxocarbenium-ion-like intermediate can be found in our simulation.