Following the Fate of Lytic Polysaccharide Monooxygenases under Oxidative Conditions by NMR Spectroscopy

Christensen, Idd Andrea; Eijsink, Vincent G. H.; Stepnov, Anton A.; Courtade, Gastón; Aachmann, Finn Lillelund

doi:10.1021/acs.biochem.3c00089

Cited by 3 publications

(1 citation statement)

References 74 publications

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“…As described in the Figure S14 , such oxygen adducts can be due to the occurrence of oxygen radicals resulting from a variety of side reactions taking place during CTZ oxidation. The situation may be akin to lytic polysaccharide monooxygenase which under certain reaction conditions is also subject to auto‐oxidation, resulting in highly heterogeneous species with low dispersion in NMR spectra (Christensen et al, 2023 ). In general, oxygen radicals give rise to addition of only one oxygen atom (Juan et al, 2021 ).…”

Section: Resultsmentioning

confidence: 99%

A suicidal and extensively disordered luciferase with a bright luminescence

Dijkema,

Escarpizo‐Lorenzana,

Nordentoft

et al. 2024

Protein Science

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Gaussia luciferase (GLuc) is one of the most luminescent luciferases known and is widely used as a reporter in biochemistry and cell biology. During catalysis, GLuc undergoes inactivation by irreversible covalent modification. The mechanism by which GLuc generates luminescence and how it becomes inactivated are however not known. Here, we show that GLuc unlike other enzymes has an extensively disordered structure with a minimal hydrophobic core and no apparent binding pocket for the main substrate, coelenterazine. From an alanine scan, we identified two Arg residues required for light production. These residues separated with an average of about 22 Å and a major structural rearrangement is required if they are to interact with the substrate simultaneously. We furthermore show that in addition to coelenterazine, GLuc also can oxidize furimazine, however, in this case without production of light. Both substrates result in the formation of adducts with the enzyme, which eventually leads to enzyme inactivation. Our results demonstrate that a rigid protein structure and substrate‐binding site are no prerequisites for high enzymatic activity and specificity. In addition to the increased understanding of enzymes in general, the findings will facilitate future improvement of GLuc as a reporter luciferase.

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Section: Resultsmentioning

confidence: 99%

A suicidal and extensively disordered luciferase with a bright luminescence

Dijkema,

Escarpizo‐Lorenzana,

Nordentoft

et al. 2024

Protein Science

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The upsurge of lytic polysaccharide monooxygenases in biomass deconstruction: characteristic functions and sustainable applications

Kumar,

Singh,

Sharma

et al. 2024

The FEBS Journal

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Lytic polysaccharide monooxygenases (LPMOs) are one of the emerging classes of copper metalloenzymes that have received considerable attention due to their ability to boost the enzymatic conversion of intractable polysaccharides such as plant cell walls and chitin polymers. LPMOs catalyze the oxidative cleavage of β‐1,4‐glycosidic bonds using molecular O2 or H2O2 in the presence of an external electron donor. LPMOs have been classified as an auxiliary active (AA) class of enzymes and, further based on substrate specificity, divided into eight families. Until now, multiple LPMOs from AA9 and AA10 families, mostly from microbial sources, have been investigated; the exact mechanism and structure–function are elusive to date, and recently discovered AA families of LPMOs are just scratched. This review highlights the origin and discovery of the enzyme, nomenclature, three‐dimensional protein structure, substrate specificity, copper‐dependent reaction mechanism, and different techniques used to determine the product formation through analytical and biochemical methods. Moreover, the diverse functions of proteins in various biological activities such as plant–pathogen/pest interactions, cell wall remodeling, antibiotic sensitivity of biofilms, and production of nanocellulose along with certain obstacles in deconstructing the complex polysaccharides have also been summarized, while highlighting the innovative and creative ways to overcome the limitations of LPMOs in hydrolyzing the biomass.

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Assessing the role of redox partners in TthLPMO9G and its mutants: focus on H2O2 production and interaction with cellulose

Chorozian,

Karnaouri,

Georgaki-Kondyli

et al. 2024

Biotechnol Biofuels

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Background The field of enzymology has been profoundly transformed by the discovery of lytic polysaccharide monooxygenases (LPMOs). LPMOs hold a unique role in the natural breakdown of recalcitrant polymers like cellulose and chitin. They are characterized by a “histidine brace” in their active site, known to operate via an O2/H2O2 mechanism and require an electron source for catalytic activity. Although significant research has been conducted in the field, the relationship between these enzymes, their electron donors, and H2O2 production remains complex and multifaceted. Results This study examines TthLPMO9G activity, focusing on its interactions with various electron donors, H2O2, and cellulose substrate interactions. Moreover, the introduction of catalase effectively eliminates H2O2 interference, enabling an accurate evaluation of each donor’s efficacy based on electron delivery to the LPMO active site. The introduction of catalase enhances TthLPMO9G’s catalytic efficiency, leading to increased cellulose oxidation. The current study provides deeper insights into specific point mutations, illuminating the crucial role of the second coordination sphere histidine at position 140. Significantly, the H140A mutation not only impacted the enzyme’s ability to oxidize cellulose, but also altered its interaction with H2O2. This change was manifested in the observed decrease in both oxidase and peroxidase activities. Furthermore, the S28A substitution, selected for potential engagement within the His1–electron donor–cellulose interaction triad, displayed electron donor-dependent alterations in cellulose product patterns. Conclusion The interaction of an LPMO with H2O2, electron donors, and cellulose substrate, alongside the impact of catalase, offers deep insights into the intricate interactions occurring at the molecular level within the enzyme. Through rational alterations and substitutions that affect both the first and second coordination spheres of the active site, this study illuminates the enzyme’s function. These insights enhance our understanding of the enzyme’s mechanisms, providing valuable guidance for future research and potential applications in enzymology and biochemistry.

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Following the Fate of Lytic Polysaccharide Monooxygenases under Oxidative Conditions by NMR Spectroscopy

Cited by 3 publications

References 74 publications

A suicidal and extensively disordered luciferase with a bright luminescence

A suicidal and extensively disordered luciferase with a bright luminescence

The upsurge of lytic polysaccharide monooxygenases in biomass deconstruction: characteristic functions and sustainable applications

Assessing the role of redox partners in TthLPMO9G and its mutants: focus on H2O2 production and interaction with cellulose

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