- Many fungi produce multiple lytic polysaccharide monooxygenases (LPMOs) with seemingly similar functions, but the biological reason for this multiplicity remains unknown. To address this question, here we carried out comparative structural and functional characterizations of three cellulose-active C4-oxidizing family AA9 LPMOs from the fungus Neurospora crassa, NcLPMO9A (NCU02240), NcLPMO9C (NCU02916), and NcLPMO9D (NCU01050). We solved the three-dimensional structure of copper-bound NcLPMO9A at 1.6-Å resolution and found that NcLPMO9A and NcLPMO9C, containing a CBM1 carbohydrate-binding module, bind cellulose more strongly and were less susceptible to inactivation than NcLPMO9D, which lacks a CBM.
- Concerns over the environment are a central driver for designing cell-free enzymatic cascade reactions that synthesize non–petrol-based commodity compounds. An often-suggested strategy that would demonstrate the economic competitiveness of this technology is recycling of valuable enzymes through their immobilization. For this purpose, amyloid nanofibrils are an ideal scaffold to realize chemistry-free covalent enzyme immobilization on a material that offers a large surface area. However, in most instances, only single enzyme–functionalized amyloid fibrils have so far been studied.
- β-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 β-glucosidases from Hypocrea jecorina, HjCel3A and HjCel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations.
- For decades, the enzymes of the fungus Hypocrea jecorina have served as a model system for the breakdown of cellulose. Three-dimensional structures for almost all H. jecorina cellulose-degrading enzymes are available, except for HjLPMO9A, belonging to the AA9 family of lytic polysaccharide monooxygenases (LPMOs). These enzymes enhance the hydrolytic activity of cellulases and are essential for cost-efficient conversion of lignocellulosic biomass. Here, using structural and spectroscopic analyses, we found that native HjLPMO9A contains a catalytic domain and a family-1 carbohydrate-binding module (CBM1) connected via a linker sequence.
- Secreted mixtures of Hypocrea jecorina cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. H. jecorina Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C. Enhanced thermal stability is desirable to enable the use of higher processing temperatures and to improve the economic feasibility of industrial biomass conversion.
- Background: The recently discovered lytic polysaccharide monooxygenases (LPMOs) are important in enzymatic conversion of lignocellulosic biomass.Results: We describe structural and functional studies of NcLPMO9C, which cleaves both cellulose and certain hemicelluloses.Conclusion: NcLPMO9C has structural and functional features that correlate with the enzyme's catalytic capabilities.Significance: This study shows how LPMO active sites are tailored to varying functionalities and adds to a growing LPMO knowledge base.