Abstract
The reaction mechanism of biomass decomposition by xylanases remains 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 crystal structure, which revealed the protonation states of relevant residues, we used ab initio quantum mechanics/molecular mechanics (QM/MM) calculations to determine the detailed reaction mechanism of the glycosylation step. 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 within 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) and 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 enzyme-substrate (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 orient residues that take part in the initial proton transfer. Because of a fine alignment of catalytic residues, the enzyme can accelerate the glycosylation reaction without paying a reorganization energy penalty.
Original language | English |
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Pages (from-to) | 17966-17980 |
Number of pages | 15 |
Journal | Journal of the American Chemical Society |
Volume | 142 |
Issue number | 42 |
DOIs | |
State | Published - Oct 21 2020 |
Funding
This international collaborative work was supported by a Fellowship grant from the Materials and Chemistry Department at the National Institute of Advanced Industrial Science and Technology (AIST). This work was supported by Grant-in-Aid for Scientific Research (C) from MEXT, KAKENHI (nos. 18K05052 and 26410031). Toyokazu Ishida is grateful to Dr. Andrey Kovalevsky for valuable discussion about the neutron structure of GH11 xylanase. J.C.S. was supported by the Genomic Science Program, Office of Biological and Environmental Research, United States Department of Energy (DOE), under Contract FWPERKP752. Oak Ridge National Laboratory is managed by UT-Battelle, LLC, for the U.S. DOE under Contract DE-AC05-00OR22725.