Amino Acid Residues Controlling Domain Interaction and Interdomain Electron Transfer in Cellobiose Dehydrogenase.

The function of cellobiose dehydrogenase (CDH) in biosensors, biofuel cells, and as a physiological redox partner of lytic polysaccharide monooxygenase (LPMO) is based on its role as an electron donor. Before donating electrons to LPMO or electrodes, an interdomain electron transfer from the catalytic FAD-containing dehydrogenase domain to the electron shuttling cytochrome domain of CDH is required. This study investigates the role of two crucial amino acids located at the dehydrogenase domain on domain interaction and interdomain electron transfer by structure-based engineering. The electron transfer kinetics of wild-type Myriococcum thermophilum CDH and its variants M309A, R698S, and M309A/R698S were analyzed by stopped-flow spectrophotometry and structural effects were studied by small-angle X-ray scattering. The data show that R698 is essential to pull the cytochrome domain close to the dehydrogenase domain and orient the heme propionate group towards the FAD, while M309 is an integral part of the electron transfer pathway - its mutation reducing the interdomain electron transfer 10-fold. Structural models and molecular dynamics simulations pinpoint the action of these two residues on the domain interaction and interdomain electron transfer.

Enzymatic Pretreatment of Plant Cells for Oil Extraction.

Oil from oilseeds can be extracted by mechanical extraction (pressing), aqueous extraction, or by extraction with organic solvents. Although solvent extraction is the most efficient method, organic solvents are a potential hazard to the life and health for workers as well as to the environment, when solvent vapours are released and act as air pollutant with a high ozone-forming potential. Pressing is safer, environmentally friendly, and it preserves valuable natural components in the resulting oils. The problems associated with pressing are the high energy consumption and the lower yield of oil extraction, because the applied mechanical force does not completely destroy the structural cell components storing the oil. In seed cells, the oil is contained in the form of lipid bodies (oleosomes) that are surrounded by a phospholipid monolayer with a protein layer on the surface. These lipid bodies are further protected by the seed cell walls consisting mainly of polysaccharides such as pectins, hemicelluloses and cellulose, but also of glycoproteins. The use of hydrolases to degrade these barriers is a promising pretreatment strategy to support mechanical extraction and improve the oil yield. It is advisable to use a combination of enzymes with different activities when considering the multicompartment and multicomponent structure of oilseed cells. This article gives an overview of the microstructure and composition of oilseed cells, reviews enzymes capable of destroying oil containing cell compartments, and summarizes the main parameters of enzymatic treatment procedures, such as the composition of the enzyme cocktail, the amount of enzyme and water used, temperature, pH, and the duration of the treatment. Finally, it analyzes the efficiency of proteolytic, cellulolytic and pectolytic enzyme pretreatment to increase the yield of mechanically extracted oil from various types of vegetable raw materials with the main focus on oilseeds.

Insights into the H2 O2 -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2 O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2 O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2 O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2 O2 -driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H2 O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2 O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2 O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions.