ORNL researchers use neutron crystallography to gain better understanding of biomass hydrolysis enzyme xylanase
Researchers led by a team from the US Department of Energy’s Oak Ridge National Laboratory (ORNL) are using neutron crystallography to understand the functioning of enzymes at the molecular level and to learn how to bioengineer those enzymes for large-scale improvements in the efficiency of biomass processing.
Using the MaNDi (Macromolecular Neutron Diffractometer) instrument at ORNL’s Spallation Neutron Source (a DOE Office of Science User Facility) (earlier post), the LANSCE (Los Alamos Neutron Science Center) Protein Crystallography Station in Los Alamos, N.M., and the FRMII BioDiff (Diffractometer for large unit cells) instrument in Munich, Germany, they determined the structure of xylanase, an enzyme used to digest hemicellulose during biofuel production, at unprecedented detail.
The work is published in the Proceedings of the National Academy of Sciences, and is the first user publication of the MaNDi instrument, which was commissioned at SNS in 2014.
Most enzymatic reactions involve hydrogen or proton transfer among the enzyme, substrate, and water at physiological pH. Thus, enzyme catalysis cannot be fully understood without accurate mapping of hydrogen atom positions in these macromolecular catalysts. Direct information on the location of hydrogen atoms can be obtained using neutron crystallography. We used neutron crystallography and biomolecular simulation to characterize the initial stage of the glycoside hydrolysis reaction catalyzed by a family 11 glycoside hydrolase. We provide evidence that the catalytic glutamate residue alternates between two conformations bearing different basicities, first to obtain a proton from the bulk solvent, and then to deliver it to the glycosidic oxygen to initiate the hydrolysis reaction.—Wan et al.
When processing plant-based biomass, hemicellulose—an abundant polysaccharide in plant cell walls—must first be degraded to monomeric sugars that can be converted to high-value products such as biofuels. Current non-harsh methods to pretreat biomass result in very basic (high pH) conditions.
Native enzymes are not very efficient in such conditions, however, preferring an acidic (low pH) environment for maximum activity. By re-engineering hemicellulose-hydrolyzing enzymes to increase their activity at high pH, researchers can improve the process, but that requires researchers to understand the intricate details of how the enzymes work.
We need to look deeper into their structures than what X-rays usually can provide. That is, we have to know where all of the hydrogen atoms are before, during and after a chemical reaction has occurred in an enzyme’s active site. Neutrons can give us this information.—ORNL’s Andrey Kovalevsky, senior author
Using neutrons, the team directly and unequivocally visualized hydrogen atoms and hydrogen bonding in xylanase at different stages of the catalytic reaction.
No one has ever observed hydrogen atoms in a glycoside hydrolase enzyme, and until now we did not know how the catalytic glutamic acid residue is protonated.—Andrey Kovalevsky
Kovalevsky and his colleagues are interested in protonation because they need to know how protons move during catalysis. For example, to start the hemicellulose hydrolysis reaction, the catalytic glutamic acid must be protonated and the catalytic base must be deprotonated. Understanding the acid/base chemistry of enzymatic biomass hydrolysis is key to rationally engineering enzymes that improve biomass processing.
Kovalevsky and his colleagues determined five neutron structures of xylanase at various pH values and in complex with a ligand. The structures showed how hydrogen atoms are arranged in the active site of xylanase, where they move and how hydrogen bonding is altered due to pH changes and ligand binding. The low-pH structure, obtained from data collected on MaNDi, helped them understand how the enzyme functions.
This enzyme, used in biofuels production, is a target for enzyme design to improve its performance in an industrial setting. Exact knowledge of its mechanism will improve protein engineering efforts.—Andrey Kovalevsky
|Nuclear density maps (green mesh) of the enzyme active site showing protonation of the catalytic glutamate residue only in the downward conformation (red mesh). Click to enlarge.|
The team has discovered that the catalytic glutamic acid can orient itself in two different conformations that have very different affinities for a proton. When the glutamate side chain rotates down and away from a substrate, it is a weaker acid than when it adopts an upward orientation. As a result, the catalytic group obtains a proton from water only when it faces downward, but can be an efficient proton donor to the substrate to initiate the hydrolysis reaction when it is in the upward conformation.
This is a big revelation for glycoside hydrolases, and specifically for xylanases, because we now know where to make amino acid substitutions in order to improve the enzyme.—Andrey Kovalevsky
The combination of neutron diffraction experiments with high-performance computing is a powerful approach for understanding how enzymes function. The researchers were curious to know how easily the glutamate side chain switches conformations. To answer that question, they turned to computer simulations.
Using neutron structures as a starting point for molecular dynamics simulations, we showed that the glutamate can readily cycle between the two conformations. With another computational approach, we also found that the acidity of the glutamate changes significantly based on how it is oriented, which agrees nicely with the neutron structures.—Jerry Parks, co-author
The research was supported by DOE’s Office of Science.
Qun Wan, Jerry M. Parks, B. Leif Hanson, Suzanne Zoe Fisher, Andreas Ostermann, Tobias E. Schrader, David E. Graham, Leighton Coates, Paul Langan, and Andrey Kovalevsky (2015) “Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography” PNAS 112 (40) 12384-12389 doi: 10.1073/pnas.1504986112