Researchers from NMBU have published an article in Nature Communications about lytic polysaccharide monooxygenases (LPMO). In the study, they show that LPMOs transfer holes (radicals) generated during catalysis via a network of aromatic amino acids. This process, often called "hole hopping", protects the enzyme from oxidative damage.
LPMOs were discovered at NMBU in 2010 and are today used all over the world for the enzymatic breakdown of biomass. LPMOs are very powerful, so-called redox enzymes that generate radicals during catalysis and that have great catalytic and industrial potential. The problem is that these radicals can also damage the enzyme.
In a new study, researchers from NMBU's Faculty of Chemistry, Biotechnology and Food Science (KBM) show that these enzymes have mechanisms that transfer radicals ('holes') away from the enzyme, through a network of aromatic amino acids. Such hole-hopping processes have been described for other types of redox enzymes, but the NMBU study stands out because it unravels a hole hopping route at an unprecedented level of detail.
About the study
Hole hopping in proteins entails the transfer of electron holes, which can also be seen as radicals. These holes or radicals are generated during catalysis and can propagate through amino acid side chains, mainly tyrosine and tryptophan, so that they move away from the enzyme rather than damaging the enzyme. It seems that hole-hopping mechanisms, which are difficult to study, have been fine-tuned in the course of evolution. Hole hopping is used for several purposes in nature, not only to protect enzymes, but also, for example, as part of the magnetic bird compass.
The holes generated during catalysis in LPMOs and some other redox enzymes are powerful oxidants that are used by the enzymes for controlled and specific oxidation of substrates. However, if catalysis fails or if no substrate is present, the holes can oxidize important amino acids in the enzyme. This would lead to damage and complete inactivation of the enzyme, unless there are protective hole-hopping routes to remove these holes, directing them to sites far from the enzyme's active site, to eventually be neutralized by reducing agents present in the solution outside the enzyme.
– In the article in Nature Communications, we analyze in detail such routes in LPMOs, says one of the lead authors at KBM and head of the research group PEP, Vincent Eijsink.
– Our study is special because LPMOs are so important and because we have been able to gain insight at a level of detail that no one else has been able to reach, he adds.
LPMOs are able to selectively activate C-H bonds in the crystalline parts of chitin and cellulose, two of the most abundant biopolymers on Earth. Breaking down the crystallinity of such polymers makes them more accessible to "ordinary" enzymes, so-called hydrolases, which leads to increased breakdown of biomass in nature and increased saccharification yields for the biomass refining industry.
LPMOs are small proteins (around 200 amino acids) with a characteristic flat surface, containing a single bound copper atom that is central to catalysis. During catalysis, hydrogen peroxide is converted into a hydroxyl radical, or a copper-oxyl radical, which represent the first hole and are powerful oxidants. If the substrate is bound, these powerful oxidants will oxidize C-H bonds in the substrate. In the absence of substrate, however, they will be able to damage amino acids near the copper and thus inactivate the enzyme.
– By using a bacterial chitin-active LPMO as a model, we have analyzed in detail how hole hopping contributes to the protection of this important protein family. Furthermore, we have shown that hole hopping can affect catalysis, because it can sometimes compete with the productive reaction, i.e., the oxidation of the substrate, says Eijsink.
He adds that this unique study has only been possible thanks to long-term funding from the ERC (European Research Council) as well as significant equipment investments by both NMBU and the Norwegian Research Council's INFRA program in the past decade.
Key results
Overall, the study shows that LPMOs have hole-hopping routes that are used for protection. The study also shows that there are different types of such routes and that some LPMOs are better protected than others, which is important when it comes to the industrial application of these unique enzymes.
The observation that activity and protection can be competing processes is the first experimental evidence for a previously hypothesized delicate balance between redox activity and redox stability in redox enzymes.