Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are recognized as important contributors to aerobic enzymatic degradation of recalcitrant polysaccharides such as chitin and cellulose. LPMOs are remarkably abundant in nature, with some fungal species possessing more than 50 LPMO genes, and the biological implications of this diversity remain enigmatic. For example, chitin-active LPMOs have been encountered in biological niches where chitin conversion does not seem to take place. We have carried out an in-depth kinetic characterization of a putatively chitin-active LPMO from Aspergillus fumigatus (AfAA11B), which, as we show here, has multiple unusual properties, such as a low redox potential and high oxidase activity. Furthermore, AfAA11B is hardly active on chitin, while being very active on soluble oligomers of N-acetylglucosamine. In the presence of chitotetraose, the enzyme can withstand considerable amounts of H₂O₂, which it uses to efficiently and stoichiometrically convert this substrate. The unique properties of AfAA11B allowed experiments showing that it is a strict peroxygenase and does not catalyze a monooxygenase reaction. This study shows that nature uses LPMOs for breaking glycosidic bonds in non-polymeric substrates in reactions that depend on H₂O₂. The quest for the true substrates of these enzymes, possibly carbohydrates in the cell wall of the fungus or its competitors, will be of major interest.

The copper-dependent lytic polysaccharide monooxygenases (LPMOs) are receiving attention because of their role in the degradation of recalcitrant biomass and their intriguing catalytic properties. The fundamentals of LPMO catalysis remain somewhat enigmatic as the LPMO reaction is affected by a multitude of LPMO- and co-substrate-mediated (side) reactions that result in a complex reaction network. We have performed kinetic studies with two LPMOs that are active on soluble substrates, NcAA9C and LsAA9A, using various reductants typically employed for LPMO activation. Studies with NcAA9C under “monooxygenase” conditions showed that the impact of the reductant on catalytic activity is correlated with the hydrogen peroxide-generating ability of the LPMO-reductant combination, supporting the idea that a peroxygenase reaction is taking place. Indeed, the apparent monooxygenase reaction could be inhibited by a competing H₂O₂-consuming enzyme. Interestingly, these fungal AA9-type LPMOs were found to have higher oxidase activity than bacterial AA10-type LPMOs. Kinetic analysis of the peroxygenase activity of NcAA9C on cellopentaose revealed a fast stoichiometric conversion of high amounts of H₂O₂ to oxidized carbohydrate products. A k_cat value of 124 ± 27 s^–1 at 4 °C is 20 times higher than a previously described k_cat for peroxygenase activity on an insoluble substrate (at 25 °C) and some 4 orders of magnitude higher than typical “monooxygenase” rates. Similar studies with LsAA9A revealed differences between the two enzymes but confirmed fast and specific peroxygenase activity. These results show that the catalytic site arrangement of LPMOs provides a unique scaffold for highly efficient copper redox catalysis.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H₂O₂ splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

CRISPR (clustered regularly interspaced short palindromic repeats)-based technologies are powerful, programmable tools for site-directed genome modifications. After successful adaptation and efficient use of CRISPR-Cas9 for genome engineering in methylotrophic yeast Komagataella phaffii, a broader variety of employable endonucleases was desired to increase the experimental flexibility and to provide alternatives in case there are specific legal restrictions in industrial research due to the intellectual property rights (IPRs) of third parties. MAD7, an engineered Class 2 Type V Cas nuclease, was promoted as a royalty-free alternative for academic and industrial research and developed by Inscripta (Pleasanton, CA, USA). In this study, for the first time, CRISPR-MAD7 was used for genome editing in K. phaffii with a high gene-editing rate (up to 90%), as demonstrated for the three targeted genes coding for glycerol kinase 1 (GUT1), red fluorescence protein (DsRed), and zeocin resistance gene (Sh ble). Additionally, the genome-editing efficiencies of the CRISPR-MAD7 and CRISPR-Cas9 systems were systematically compared by targeting 259 kinase genes in K. phaffii. In this broad testing, the CRISPR-Cas9 had a higher genome-editing rate of about 65%, in comparison to the applied CRISPR-MAD7 toolbox (about 23%).

Lytic polysaccharide monooxygenases (LPMOs) are important biotechnological tools due to their ability to activate C–H bonds in recalcitrant polysaccharides. To-date, most research has focused on LPMOs from the AA9 and AA10 families, while LPMOs from the AA11 family have not received the same attention since their classification almost a decade ago, despite their wide abundance in fungi. Previous studies have shown that AfAA11B from Aspergillus fumigatus has exceptionally high oxidase activity, low reduction potential and the ability to degrade soluble chitooligomers. To better understand the catalytic capabilities of AfAA11B, its crystal structure was solved, revealing a unique flexible surface loop that mediates activity on soluble substrates, as shown by molecular dynamics simulations and mutagenesis. Mutation of an active site Glu residue to a Gln, Asp or Asn showed that this residue is crucial in controlling the low reduction potential and high oxidase activity of AfAA11B. The impact of these mutations on copper reactivity aligned well with results obtained for an AA9 LPMO, which naturally has a Gln in this position. However, the impact of these mutations on the productive peroxygenase reaction, measured using an electrochemical hydrogen peroxide sensor, and on protective hole hopping mechanisms, measured using stopped-flow ultraviolet–visible (UV–vis) spectrophotometry, differed from the AA9 LPMO. This shows that the impact of this Glu/Gln residue is dependent on additional structural or dynamic differences between the LPMOs. Despite the presence of several tryptophan residues in the protein core, the hole hopping studies revealed formation of only a tyrosyl feature with a lifespan distinct from similar features detected in other LPMOs, further highlighting the unique properties of AfAA11B.