Mechanism, function and diversity of a novel family of biomass degrading enzymes

The project "Mechanism, function and diversity of a novel family of biomass degrading enzymes" has explored the properties of a recently discovered polysaccharide degrading enzyme family using a "basic research"-type approach. The reaction catalyzed by this new enzyme activity is the cleavage of the glycosidic bonds of carbohydrates by an oxidative mechanism. The catalyzed reaction has given the enzyme family its name; "lytic polysaccharide monooxygenase" (LPMO). "Lytic" indicates that the enzyme cleaves something, polysaccharide indicates the substrate and "monooxygenase" describes the reaction mechanism where a single oxygen atom is introduced on the final product of the reaction. The unique property of the LPMOs is their ability to perform oxidative chemistry on the surface of insoluble, crystalline polysaccharides such as cellulose and chitin. This reaction leads to disruption of the fiber surface, which yields it more accessible for enzymes that requires access to "free" chains to work. LPMOs and their enzyme activity were discovered by the project leader five years ago in Science magazine, a discovery that today is acknowledged as an important contribution to the development of enzyme technology designed for efficient solubilization of recalcitrant biomass. The project, which has been a collaboration between NMBU (Gustav Vaaje-Kolstad) and NTNU (Finn L. Aachmann), lasted for three years and had fundamental exploration of the LPMO reaction as its main goal. The project had a flying start with a publication in the internationally renown journal PNAS (Proceedings of the National Academy of Science of the USA; Aachmann et al. 2012). This study explored the dynamic, redox and copper binding properties of CBP21, a LPMO from the soil bacterium Serratia marcescens. The majority of the analysis work performed in this study was performed using nuclear magnetic resonance spectroscopy (NMR), the special competence of the NTNU partner. On a general basis the results emerging from the study yielded a deeper understanding of the fundamental properties of LPMOs and also generated new ideas for how to further explore this enzyme family. A technique that is complementary to NMR is electron paramagnetic resonance spectroscopy (EPR). In a collaboration with scientists from UiO we used this technique combined with classical biochemical techniques to study the active site copper-center of several diverse LPMOs. An important finding in this study was that LPMOs with difference substrate preferences (cellulose or chitin) showed essentially identical catalytic centers, but had different EPR signatures (Forsberg et al., Biochemistry 2014). This indicates that the LPMO substrate specificity is dictated by residues located outside the catalytic center, which also influence the EPR properties of the enzyme. One of the project highlights was achieved by comparing two LPMOs that both degrade cellulose, but with subtly different reaction mechanisms; either oxidation of carbon 1 (C1) or carbon 4 (C4) of the sugar chain (Forsberg et al. PNAS 2014). When the two enzymes were mixed with cellulose, a substantial synergy in substrate degradation was observed. This was the first time a synergy between two LPMOs has been shown. The results indicated that the enzymes have complementary properties that have been conserved during evolution. Furthermore, the three-dimensional structures of the enzyme pair was solved, which gave a deeper insight into how these LPMOs catalyze oxidation of cellulose, especially the properties that determine oxidation of either C1 or C4. In the final stage of the project we also conducted studies on important virulence factors (proteins involved in the process of bacterial infection) that resemble LPMOs, but that have an unknown function. In two separate studies we were able to demonstrate that such virulence factors from the notorious pathogens Vibrio cholera and Listeria monocytogenes had LPMO activity (i.e. were able to cleave polysaccharides; Loose et al., FEBS Letters 2014 and Paspaliari et al. FEBS Journal 2015). These are important findings that may change the understanding of how bacteria infect their hosts. Overall, we can conclude that the project has successfully achieved its goals. We have generated a substantial amount of new knowledge on how LPMOs cleave sugar chains and how these enzymes may contribute to virulence and bacterial infections. The project has strengthened the collaboration between NTNU and NMBU and has provided new ideas and inspired several projects that will stimulate and develop future research on LPMOs, something that will benefit both science and society.

The enzymatic conversion of polysaccharides plays crucial roles in processes as diverse as microbial food scavenging, cellular development in plants and fungi, and host-pathogen interactions. On the industrial side, the development of the future biorefine ry depends critically on the development of effective enzyme technology for the conversion of recalcitrant polysaccharides such as chitin and, especially, cellulose. The recent discovery of a novel class of polysaccharide-degrading enzymes currently class ified as "CBM33" and "GH61" (Vaaje-Kolstad et al., 2010, Science 330:219-222) has opened new avenues for more effective enzymatic biomass processing and raised many novel questions concerning how enzymatic conversion of polysaccharides takes place in natu ral settings. These metal- and oxygen-dependent enzymes use an unprecedented catalytic mechanism. Furthermore they occur in multiple copies per organism and in a wide variety of ecological niches, suggesting variation in substrate specificities and in viv o functionalities. Considering their function and their abundance, the discovery of these novel proteins calls for a dedicated effort to unravel their catalytic mechanisms as well as their functional diversity and the structural basis thereof. In this pro ject we will sample CBM33 diversity by cloning, expression, purification and characterization efforts. Pure enzymes and their individual domains will be subjected to a wide array of biochemical characterization methods, in part based on the applicant's top competence in the analysis of complex carbohydrates. To generate deeper insight into structure and function, we will use characterization techniques such as electron paramagnetic resonance (for mechanistic studies), NMR (structure, dynamics, pKa titra tions, metal binding) and X-ray crystallography (structure). As new knowledge on the function and specificities of the enzymes accumulates, their in vivo functions will be explored in more detail.