Electron flow during oxidative cellulose degradation

Plant biomass represents the largest terrestrial carbohydrate resource. Wood-degrading fungi utilize this vast energy reservoir by secreting specific enzymes which degrade recalcitrant biopolymers in wood into digestible sugars. A recently discovered enzyme system occupies a central role in this process: It loosens the dense structures in the biomass and enables its degradation by other enzymes. So-called lytic polysaccharide monooxygenases (LPMOs) are key players in this process. They target highly recalcitrant, crystalline parts of the substrate. However, LPMOs are not self- sufficient enzymes, but require helper enzymes, so-called cellobiose dehydrogenases (CDHs), for full activity. A key mechanism in this activation process is a directed electron transport chain, which triggers LPMO catalysis. This electron flow proceeds through several reactive centres in CDH and LPMO, and requires a high flexibility and dynamic of these enzymes. The aim of this project is to describe this dynamic behaviour with a set of methods having a high temporal and special resolution. This goal shall be achieved in a collaborative project involving the research groups of Dr. Roland Ludwig (BOKU University, Vienna) and Prof. Nigel Scrutton (University of Manchester). Using highly specialized equipment in Manchester enables to resolve and investigate single enzymatic reaction steps and the underlying protein motions with a temporal resolution down to the pico-second range. In Vienna, a complementary approach, which allows locating enzymes on surfaces, will be used to study the interaction and distribution of CDH and LPMO on their natural substrate, cellulose. The basis of this approach is an electrochemical electron microscope, which allows detecting enzymes based on their catalytic activity. A general understanding of the activity and interaction of these enzymes is crucial to understand fungal physiology, but is also the conditio sine qua non for their application, e.g. to transform plant biomass into energy or renewable hydrocarbons.

Biomass-degrading fungi occupy a central role in the terrestrial carbon cycle. Their remarkable ability to degrade and assimilate the most recalcitrant components of plant cell walls, such as cellulose, is based on a set of highly specialized enzymes. A recently discovered class of cellulose-degrading enzymes was shown to greatly contribute to this process. Activation of these enzymes depends on the coordinated transfer of electrons, which proceeds through multiple reactive centres and involves highly dynamic protein motions. In this research project, these electron transfer reactions were measured with high temporal resolution and were correlated with the underlying dynamic protein motions. A broad spectrum of techniques, involving mostly biochemical but also molecular biological and computer-aided methods, were used to unravel the interaction of the investigated enzymes on a molecular level. This goal was achieved in a collaborative project involving the research groups of Dr Roland Ludwig (BOKU University, Vienna) and Prof. Nigel Scrutton (University of Manchester). Furthermore, a new method was developed that allows measuring the degradation of cellulose by these enzymes in real-time and without laborious and time-consuming analysis methods. Results obtained during this project, therefore, not only help to improve our understanding of fungal physiology but are also important for the application of cellulose-degrading enzymes to produce energy or value-added chemicals from renewable resources such as plant biomass.