| dc.description.abstract | Global warming and the pending energy crisis pressure the search for effective utilization of renewable energy sources. Biomass has long been discussed to be such an energy source, but the recalcitrant constituents, such as cellulose, hemicellulose and lignin, make the utilization of the full potential hard to reach. Several pretreatments of plant biomass have been developed to increase the degradation; however, these are either expensive or time-consuming, and therefore the use of biomass is not very effective with today’s technologies. Lytic polysaccharide monooxygenases (LPMOs) cleave glycosidic bonds, and can be active on crystalline polysaccharide structures, where they introduce chain breaks achieving an increase in oxidized ends for other carbohydrate active enzymes to act on, ensuring a sustainable enzymatic catalyzed solubilization of, i.e., cellulose and chitin, which are biopolymers of abundance. LPMOs are metalloenzymes, with a copper ion coordinated by two histidines in the active site; a configuration called the histidine brace. A reductant is needed to activate the LPMO by reducing the copper, whereafter the LPMO, along with a co-substrate that is most likely H2O2, oxidizes either the C1 or C4 of the glycosidic bond in the substrate. Like other oxidoreductases, LPMOs are prone to oxidative damage and the enzyme inactivation that can follow. Understanding the reaction mechanism and factors that potentially lead to enzyme inactivation is industrially relevant for optimizing LPMOs for carbohydrate degradation.
This MSc thesis investigates the role of two post-translational modifications (PTMs) (glycosylation and methylation) on LPMO activity and protection. Two fungal LPMOs from the AA9 family found in Thermoascus aurantiacus (TtAA9E) and Thielavia terrestris (TaAA9A) were produced in two different expression hosts: one capable of performing the post-translational methylation (Aspergillus oryzae) and the other not (Pichia pastoris). The two TtAA9E variants were deglycosylated and the four enzymes were subjected to activity experiments. Very similar oxidase activity and enzyme activity on PASC under monooxygenase and peroxygenase was observed for all TtAA9E variants, suggesting that glycosylation and methylation have little effect on the LPMO catalytic mechanism. It is noteworthy that TtAA9E required twice the concentration of exogenous H2O2 to be inactivated, compared to TaAA9A. Besides enzyme activity experiments, the transient-state kinetics of methylated and non-methylated TtAA9E and TaAA9A were compared, to determine possible changes in the rate of reduction and reoxidation of the active-site copper between the LPMO variants. Interestingly, non-methylated TaAA9A showed an increase in the reduction rate, but a very similar reoxidation rate compared to the methylated variant. Both reduction and reoxidation rates of methylated and non-methylated TtAA9E were very similar, suggesting an unchanged enzyme mechanism. Lastly, the radical formation of Tyr• and Trp• was monitored, aiming to achieve information about amino acid residues linked to the protective hole-hopping reaction. Both TtAA9E variants showed no Tyr• and Trp• formation. The estimated Trp• formation was twice as high in the methylated TaAA9A compared to the non-methylated variant, suggesting a possible increase in protecting hole-hopping activity for the methylated LPMO.
The work presented here shows that the role of glycosylation on enzyme activity is insignificant in TtAA9E. It also shows that methylation has a protecting role against oxidative damage in TaAA9A, possibly by engaging a protective hole-hopping pathway, and in that way removing the potentially damaging oxidizing equivalent away from the active site. In addition, despite TtAA9E having multiple Tyr residues near the active site copper and having a Trp residue located at the same location as TaAA9A, these residues do not participate in hole-hopping. These results compel for further investigation into the role of methylation in TtAA9E and other fungal AA9. | |
