Electron Transfer in Cellulose Degrading Enzymes

The interaction of the two enzymes lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH) was shown to boost the effectiveness of cellulose degradation. While CDH has been proposed to act as a reductant for LPMO, its specific action is unclear. To investigate this extracellular redox process and its relevance in comparison with other potential reductants such as gallic acid, we will employ CDH and LPMO enzymes from cellulose degrading fungi. The interaction of LPMO and CDH in homogeneous solution and more important the physiologically relevant state of cellulose bound enzymes will be characterized. Stopped-flow spectrophotometry and isothermal calorimetry will be used to elucidate electron transfer rates and protein interactions. To study the kinetics of bound enzymes on cellulosic substrates we will employ steady-state kinetic analysis based on HPLC, mass spectrometry analysis of reaction products. Protein cross-linking followed by structural mass-spectrometry and proton exchange experiments will be used to elucidate protein-protein and protein-cellulose interactions. The project will test the hypothesis that CDHs natural role is that of an LPMO reductase. The CDH/LPMO interaction will be studied to specifically answer three questions: 1) Is CDH a fast electron donor for LPMO and how does it compete with small molecular mass reductants such as gallic acid? 2) Do CDH and LPMO interact by a specific protein-protein interface or is the interprotein electron transfer based on random contacts? 3) Differs the interaction of CDHs and LPMOs with a carbohydrate binding module from the ones which do not? The results of this study will expand the understanding of extracellular, biocatalytic redox processes as the basis of oxidative celllulose degradation. Applied research will benefit from the approaches to modulate and engineer the interaction of CDH and LPMO, which can lead to an increased efficiency of second generation biofuel production or depolymerization processes in green biorefineries.

The interaction of the two enzymes lytic polysaccharide monooxygenase (LPMO) and cellobiose dehydrogenase (CDH) was shown to boost the effectiveness of cellulose degradation. While CDH has been proposed to act as a reductant for LPMO, its specific action has been unclear and was investigated in this project. It was found that CDH does not only provide electrons to activate LPMO by reducing its copper center, but also supplies hydrogen peroxide as a cosubstrate. The interaction of both enzymes has been modeled and verified by mutagenesis experiments. This extracellular redox process has been compared with other potential reductants such as gallic acid for a number of CDH and LPMO enzymes from cellulose degrading fungi. The interaction of LPMO and CDH in homogeneous solution and more important the physiologically relevant state of cellulose bound enzymes was characterized. Stopped-flow spectrophotometry and isothermal calorimetry were used to elucidate electron transfer rates and protein interactions. To study the kinetics of bound enzymes on cellulosic substrates we employed steady-state kinetic analysis based on HPLC, mass spectrometry analysis of reaction products. Protein cross-linking followed by structural mass-spectrometry and proton exchange experiments will be used to elucidate protein-protein and protein-cellulose interactions. The project found that CDH's natural role is indeed that of an "LPMO reductase". The CDH/LPMO interaction occurs at the interface to the protein cofactors at a specific protein-protein interface. The interaction of CDHs and LPMOs are modulated by the presence of a carbohydrate binding (CBM). The results of this study expand the understanding of extracellular, biocatalytic redox processes as the basis of oxidative celllulose degradation. Applied research benefits from the new approaches to modulate and engineer the interaction of CDH and LPMO, which leads to an increased efficiency of second generation biofuel production and depolymerization processes in green biorefineries.