Resolving molecular mechanisms of biomass degrading enzymes through a combined computational and experimental approach

Recalcitrant polysaccharides as cellulose and chitin consist of long chains of sugar molecules that are compactly packed together. These highly abundant biomass feedstocks are valuable resources for current and future biorefining processes, e.g. production of bioethanol, that have positive impacts on the environment and society. A major bottleneck of recalcitrant polysaccharide utilization is solubilization of the individual sugar chains, making them available for hydrolytic enzymes. The recently discovered enzymes named lytic polysaccharide monooxygenases (LPMOs), are designed by Nature to solve this problem, cleaving polysaccharide chains packed in a crystalline form. It was thought that LPMOs use molecular oxygen and copper to cleave cellulose and chitin. However, the oxygen activation mechanism and the overall reaction mechanism with substrate were not known and the initial models had several issues. In 2017, in collaboration with a NMBU research group, we found that hydrogen peroxide is preferred over molecular oxygen as oxidative co-substrate. We have identified how chitin active LPMOs bind to various forms of chitin applying molecular dynamics simulations and supporting these data using EPR spectroscopy and biochemical assays. Also, we have looked at protonation states of near active site amino acid residues of cellulose active LPMOs using molecular dynamics simulations, obtaining data important for further studies of the LPMO reaction mechanism. In 2018, we have taken advantage of rapid kinetics methods, dissecting the LPMO reaction mechanism by experiment. When combining these novel results with computational data, we are now able to understand how this enzyme reacts with both oxygen and hydrogen peroxide, contributing to explain several controversial observations made by other groups. The synergetic effects of combining the selected methods have contributed to a better understanding of oxidative enzymes like LPMOs. With detailed knowledge about the LPMO reaction mechanism, we can start to modify the enzymatic activity and specificity of these enzymes. In the future, the unique catalytic properties of LPMOs may be utilized to modify a number of substrates, expanding the industrial potential for these enzymes.

Dette prosjektet har involvert og hatt virkning på en rekker personer; en stipediat, en forsker og flere nasjonale og internasjonale samarbeidspartnere. Personene som har vært involvert har delt informasjon og dette har økt til økt kompetanse hos alle involverte samt etablering av ny metodikk på NMBU. Effektene fra dette grunnforskningsprosjektet ventes å slå inn på fagfeltene biokatalyse og foredling av biomasse. Vi har bidratt til å forstå hvordan enzymer fungerer, og har derfor vært med å forme grunnlaget for industrielle anvendelser.

The highly abundant biomass feedstocks of the recalcitrant polysaccharides cellulose and chitin are valuable resources for current and future biorefining processes that have positive impacts on the environment and society. A major bottleneck of recalcitrant polysaccharide utilization is solubilization of the individual sugar chains, making them available for hydrolytic enzymes. The recently discovered enzymes named lytic polysaccharide monooxygenases (LPMOs), are designed by Nature to solve this problem, cleaving polysaccharide chains packed in a crystalline form. LPMOs use molecular oxygen and copper to cleave cellulose and chitin. However, the oxygen activation mechanism and the overall reaction mechanism with substrate are not known. We aim to identify those mechanisms using a combined computational and experimental approach. The synergetic effects of combining the selected methods are expected to result in massive contributions to the LPMO and biorefining research fields. With detailed knowledge about the LPMOs reaction mechanisms, revealed structural and electronic determinants associated with enzymatic activity can be used to optimize LPMOs, change their substrate specificity, and alter the oxidation products. Also included in this project, is a route of rational enzyme design that aims to combine an in silico mutagenesis approach with in vitro experiments to create more efficient LPMOs. This is pioneering work that can be translated directly into the biofuel production technologies.