Understanding the Catalytic Mechanism and the Substrate Specificity of an Engineered Gluten Hydrolase by QM/MM Molecular Dynamics and Free Energy Simulations.

Celiac sprue, also known as gluten-sensitive enteropathy, is a chronic disease suffered by approximately 1% of the world's population. Engineered enzymes have been emerging to treat celiac disease by hydrolyzing the pathogenic peptides of gluten. For example, Kuma010 has been studied experimentally and proved to be a promising gluten hydrolase under gastric conditions. However, the detailed catalytic mechanism and the substrate specificity are still unclear. In this paper, quantum-mechanical/molecular-mechanical (QM/MM) molecular dynamics (MD) and free energy simulations were performed to determine the catalytic mechanism, the substrate specificity, and the role of the active-site residues during the reaction. The results given here demonstrate that the Kuma010 has a similar catalytic mechanism but different substrate specificity as wild-type kumamolisin-As. Binding properties of the enzyme (especially mutated residues) and substrate complex are discussed, and activation free energy barriers toward different substrates have also been examined. The computational free energy results are in reasonable agreement with the experimental data. The strategy for developing next-generation gluten hydrolases is discussed.