Insight into the Catalytic Mechanism of GH11 Xylanase: Computational Analysis of Substrate Distortion based on a Neutron Structure.

The reaction mechanism of xylanase biomass decomposition enzymes has been the subject of debate. To clarify the mechanism we investigated the glycosylation step of GH11 xylanase, an enzyme that catalyzes the hydrolysis of lignocellulosic hemicellulose (xylan). Making use of a recent neutron structure, which identified the protonation states of relevant residues, we use ab initio QM/MM methodology to determine the detailed reaction mechanism of the glycosylation. In particular, our focus is on the controversial question of whether or not an oxocarbenium ion intermediate is formed on the reaction pathway. The calculations support the validity of a basic retaining mechanism with a double displacement scheme. The estimated free energy barrier of this reaction is ~18 kcal/mol with QM/MM-CCSD(T)/6-31(+)G**//MP2/6-31+G**/AMBER calculations, and the rate-determining step of the glycosylation is scission of the glycosidic bond after proton transfer from the acidic Glu177. The estimated lifetime of the oxocarbenium ion intermediate (on the order of tens of ps) as well as the secondary kinetic isotope effect suggest that there is no accumulation of this intermediate on the reaction path, although the intermediate can be transiently formed. In the ES complex, the carbohydrate structure of the xylose residue at the -1 subsite has a rather distorted (skewed) geometry, and this xylose unit at the active site has an apparent half-chair conformation when the oxocarbenium ion intermediate is formed. The major catalytic role of the protein environment is to appropriately fix and align residues that take part in the initial proton transfer. Due to a fine alignment of catalytic residues, the enzyme can accelerate the glycosylation reaction without paying a reorganization energy penalty.