AA16 Oxidoreductases Boost Cellulose-Active AA9 Lytic Polysaccharide Monooxygenases from Myceliophthora thermophila

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) classified in Auxiliary Activity (AA) families are considered indispensable as synergistic partners for cellulolytic enzymes to saccharify recalcitrant lignocellulosic plant biomass. In this study, we characterized two fungal oxidoreductases from the new AA16 family. We found that MtAA16A from Myceliophthora thermophila and AnAA16A from Aspergillus nidulans did not catalyze the oxidative cleavage of oligo- and polysaccharides. Indeed, the MtAA16A crystal structure showed a fairly LPMO-typical histidine brace active site, but the cellulose-acting LPMO-typical flat aromatic surface parallel to the histidine brace region was lacking. Further, we showed that both AA16 proteins are able to oxidize low-molecular-weight reductants to produce H2O2. The oxidase activity of the AA16s substantially boosted cellulose degradation by four AA9 LPMOs from M. thermophila (MtLPMO9s) but not by three AA9 LPMOs from Neurospora crassa (NcLPMO9s). The interplay with MtLPMO9s is explained by the H2O2-producing capability of the AA16s, which, in the presence of cellulose, allows the MtLPMO9s to optimally drive their peroxygenase activity. Replacement of MtAA16A by glucose oxidase (AnGOX) with the same H2O2-producing activity could only achieve less than 50% of the boosting effect achieved by MtAA16A, and earlier MtLPMO9B inactivation (6 h) was observed. To explain these results, we hypothesized that the delivery of AA16-produced H2O2 to the MtLPMO9s is facilitated by protein–protein interaction. Our findings provide new insights into the functions of copper-dependent enzymes and contribute to a further understanding of the interplay of oxidative enzymes within fungal systems to degrade lignocellulose.

[1]  M. Marletta,et al.  A moonlighting function of a chitin polysaccharide monooxygenase, CWR-1, in Neurospora crassa allorecognition , 2022, eLife.

[2]  V. Eijsink,et al.  Enhanced in situ H2O2 production explains synergy between an LPMO with a cellulose-binding domain and a single-domain LPMO , 2022, Scientific Reports.

[3]  S. Ovchinnikov,et al.  ColabFold: making protein folding accessible to all , 2022, Nature Methods.

[4]  J. Berrin,et al.  On the expansion of biological functions of Lytic Polysaccharide Monooxygenases (LPMOs). , 2021, The New phytologist.

[5]  W. V. van Berkel,et al.  Oxidized Product Profiles of AA9 Lytic Polysaccharide Monooxygenases Depend on the Type of Cellulose , 2021, ACS sustainable chemistry & engineering.

[6]  B. Henrissat,et al.  Secreted pectin monooxygenases drive plant infection by pathogenic oomycetes , 2021, Science.

[7]  Oriol Vinyals,et al.  Highly accurate protein structure prediction with AlphaFold , 2021, Nature.

[8]  V. Eijsink,et al.  Sugar oxidoreductases and LPMOs - two sides of the same polysaccharide degradation story? , 2021, Carbohydrate research.

[9]  Marco Zarattini,et al.  A fast and easy strategy for lytic polysaccharide monooxygenase-cleavable His6-Tag cloning, expression, and purification. , 2021, Enzyme and microbial technology.

[10]  G. Moore,et al.  Anomalous collapses of Nares Strait ice arches leads to enhanced export of Arctic sea ice , 2021, Nature communications.

[11]  B. Henrissat,et al.  Discovery of fungal oligosaccharide-oxidising flavo-enzymes with previously unknown substrates, redox-activity profiles and interplay with LPMOs , 2020, Nature Communications.

[12]  OUP accepted manuscript , 2021, Nucleic Acids Research.

[13]  J. Berrin,et al.  Evaluation of the Enzymatic Arsenal Secreted by Myceliophthora thermophila During Growth on Sugarcane Bagasse With a Focus on LPMOs , 2020, Frontiers in Bioengineering and Biotechnology.

[14]  R. Shoeman,et al.  BioMAX – the first macromolecular crystallography beamline at MAX IV Laboratory , 2020, Journal of synchrotron radiation.

[15]  O. Svensson,et al.  ID30A-3 (MASSIF-3) – a beamline for macromolecular crystallography at the ESRF with a small intense beam , 2020, Journal of synchrotron radiation.

[16]  W. V. van Berkel,et al.  Mass spectrometric fragmentation patterns discriminate C1- and C4-oxidised cello-oligosaccharides from their non-oxidised and reduced forms. , 2020, Carbohydrate polymers.

[17]  B. Henrissat,et al.  A fungal family of lytic polysaccharide monooxygenase-like copper proteins , 2020, Nature Chemical Biology.

[18]  N. Grishin,et al.  A Lytic Polysaccharide Monooxygenase-like protein functions in fungal copper import and meningitis , 2019, Nature Chemical Biology.

[19]  Liisa Holm,et al.  Using Dali for Protein Structure Comparison. , 2020, Methods in molecular biology.

[20]  C. Oostenbrink,et al.  Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity , 2019, International journal of molecular sciences.

[21]  M. Sandgren,et al.  Comparison of three seemingly similar lytic polysaccharide monooxygenases from Neurospora crassa suggests different roles in plant biomass degradation , 2019, The Journal of Biological Chemistry.

[22]  C. Rovira,et al.  Molecular Mechanisms of Oxygen Activation and Hydrogen Peroxide Formation in Lytic Polysaccharide Monooxygenases , 2019, ACS catalysis.

[23]  V. Eijsink,et al.  Lytic Polysaccharide Monooxygenases in Enzymatic Processing of Lignocellulosic Biomass , 2019, ACS Catalysis.

[24]  B. Henrissat,et al.  AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes , 2019, Biotechnology for Biofuels.

[25]  V. Eijsink,et al.  pH-Dependent Relationship between Catalytic Activity and Hydrogen Peroxide Production Shown via Characterization of a Lytic Polysaccharide Monooxygenase from Gloeophyllum trabeum , 2018, Applied and Environmental Microbiology.

[26]  V. Eijsink,et al.  Methylation of the N‐terminal histidine protects a lytic polysaccharide monooxygenase from auto‐oxidative inactivation , 2018, Protein science : a publication of the Protein Society.

[27]  V. Eijsink,et al.  Kinetics of H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase. , 2018, The Journal of Biological Chemistry.

[28]  V. Eijsink,et al.  The impact of hydrogen peroxide supply on LPMO activity and overall saccharification efficiency of a commercial cellulase cocktail , 2018, Biotechnology for Biofuels.

[29]  John A. Hangasky,et al.  Reactivity of O2 versus H2O2 with polysaccharide monooxygenases , 2018, Proceedings of the National Academy of Sciences.

[30]  Paul Emsley,et al.  Structural analysis of glycoproteins: building N-linked glycans with Coot , 2018, Acta crystallographica. Section D, Structural biology.

[31]  C. Oostenbrink,et al.  A fast and sensitive activity assay for lytic polysaccharide monooxygenase , 2018, Biotechnology for Biofuels.

[32]  B. Dalhus,et al.  Structural determinants of bacterial lytic polysaccharide monooxygenase functionality , 2017, The Journal of Biological Chemistry.

[33]  V. Eijsink,et al.  Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2. , 2017, Nature chemical biology.

[34]  J. Berrin,et al.  Fungal secretomics to probe the biological functions of lytic polysaccharide monooxygenases. , 2017, Carbohydrate research.

[35]  M. Marletta,et al.  The Role of the Secondary Coordination Sphere in a Fungal Polysaccharide Monooxygenase. , 2017, ACS chemical biology.

[36]  J. Visser,et al.  Discovery of a Xylooligosaccharide Oxidase from Myceliophthora thermophila C1* , 2016, The Journal of Biological Chemistry.

[37]  B. Svensson,et al.  Lytic polysaccharide monooxygenases and other oxidative enzymes are abundantly secreted by Aspergillus nidulans grown on different starches , 2016, Biotechnology for Biofuels.

[38]  J. Visser,et al.  Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity , 2016, Biotechnology for Biofuels.

[39]  D. Haltrich,et al.  Extracellular electron transfer systems fuel cellulose oxidative degradation , 2016, Science.

[40]  Thomas J. Simmons,et al.  The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases. , 2016, Nature chemical biology.

[41]  G C P van Zundert,et al.  The HADDOCK2.2 Web Server: User-Friendly Integrative Modeling of Biomolecular Complexes. , 2016, Journal of molecular biology.

[42]  V. Eijsink,et al.  Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions , 2015, Biotechnology for Biofuels.

[43]  J. Visser,et al.  Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase , 2015, Biotechnology for Biofuels.

[44]  M. Marletta,et al.  Cellulose degradation by polysaccharide monooxygenases. , 2015, Annual review of biochemistry.

[45]  M. Fraaije,et al.  A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase , 2014, FEBS letters.

[46]  J. Saddler,et al.  Substrate factors that influence the synergistic interaction of AA9 and cellulases during the enzymatic hydrolysis of biomass , 2014 .

[47]  A. Gronenborn,et al.  Weak protein complexes: challenging to study but essential for life , 2014, The FEBS journal.

[48]  Adrie J J Straathof,et al.  Transformation of biomass into commodity chemicals using enzymes or cells. , 2014, Chemical reviews.

[49]  H. Jørgensen,et al.  Do new cellulolytic enzyme preparations affect the industrial strategies for high solids lignocellulosic ethanol production? , 2014, Biotechnology and bioengineering.

[50]  B. Henrissat,et al.  Discovery and characterization of a new family of lytic polysaccharide mono-oxygenases , 2013, Nature chemical biology.

[51]  Pedro M. Coutinho,et al.  The carbohydrate-active enzymes database (CAZy) in 2013 , 2013, Nucleic Acids Res..

[52]  D. Haltrich,et al.  Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay , 2012, Biotechnology for Biofuels.

[53]  Jamie H. D. Cate,et al.  Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases. , 2012, Journal of the American Chemical Society.

[54]  Jamie H. D. Cate,et al.  Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. , 2011, ACS chemical biology.

[55]  L. Lo Leggio,et al.  Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components , 2011, Proceedings of the National Academy of Sciences.

[56]  Feng Xu,et al.  Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61 , 2011, Applied and Environmental Microbiology.

[57]  A. Gusakov,et al.  Development of a mature fungal technology and production platform for industrial enzymes based on a Myceliophthora thermophila isolate, previously known as Chrysosporium lucknowense C1 , 2011 .

[58]  N. Pannu,et al.  REFMAC5 for the refinement of macromolecular crystal structures , 2011, Acta crystallographica. Section D, Biological crystallography.

[59]  Francesco Cherubini,et al.  The biorefinery concept: Using biomass instead of oil for producing energy and chemicals , 2010 .

[60]  Liisa Holm,et al.  Dali server: conservation mapping in 3D , 2010, Nucleic Acids Res..

[61]  Fabrice Gorrec,et al.  The MORPHEUS protein crystallization screen , 2009, Journal of applied crystallography.

[62]  Werner Braun,et al.  InterProSurf: a web server for predicting interacting sites on protein surfaces , 2007, Bioinform..

[63]  D. Janssen,et al.  Changing the substrate specificity of a chitooligosaccharide oxidase from Fusarium graminearum by model‐inspired site‐directed mutagenesis , 2007, FEBS letters.

[64]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[65]  E. Golightly,et al.  A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale. , 2001, European journal of biochemistry.

[66]  C. Koch,et al.  Mechanism of copper-catalyzed autoxidation of cysteine. , 1999, Free radical research.

[67]  A. Vagin,et al.  MOLREP: an Automated Program for Molecular Replacement , 1997 .

[68]  A. Beezer,et al.  A kinetic study of the oxidation of L-ascorbic acid (vitamin C) in solution using an isothermal microcalorimeter , 1995 .

[69]  C. Stevens,et al.  Aquaporin 4 and glymphatic flow have central roles in brain fluid homeostasis , 2021, Nature Reviews Neuroscience.