Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

Kuusk, Silja; Kont, Riin; Kuusk, Piret; Heering, Agnes; Sørlie, Morten; Bissaro, Bastien; Eijsink, Vincent G. H.; Väljamaë, Priit

doi:10.1074/jbc.ra118.006196

Cited by 67 publications

(100 citation statements)

References 64 publications

(110 reference statements)

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“…First, we observed that the rate of NcLPMO9C increased linearly with the concentration of ascorbate. While we cannot exclude experimentally that the assays may not have been carried out under saturating ascorbate concentrations, a recent study showed that the bacterial LPMO10A from Serratia marcescens had an apparent K M -value of 2 µM for ascorbate [44]. Even if the K M -value of NcLPMO9C for ascorbate would be 50-times higher, the high 0.5-6 mM ascorbate concentration present in our assays should still provide sufficiently saturating conditions to achieve maximal turnover.…”

Section: Discussionmentioning

confidence: 90%

“…A growing body of evidence demonstrates that LPMOs use H 2 O 2 as cosubstrate with a much higher catalytic efficiency than O 2 [26,28,44,51]. While the cosubstrate preference of LPMOs in their native environments is still debated [29] the efficient peroxygenase reactivity may be beneficial in industrial settings to achieve faster biomass depolymerization [52].…”

Section: Discussionmentioning

confidence: 99%

“…4a, red The activity of LPMOs is typically assessed in the presence of an about 1 mM concentration of ascorbate, which reduces the active-site copper and initiates the oxidative degradation of the substrate. Several recent publications, however, raised the question whether the observed activity is due to an O 2 -dependent monooxygenase reaction, or, at least partially, depends on the H 2 O 2 that is slowly released by the reaction of oxygen with the reductant ascorbate [26,31,44]. In addition, reduced LPMOs in solution may also release low H 2 O 2 concentrations via an uncoupling reaction [36].…”

Section: Discussionmentioning

confidence: 99%

“…1e, black circles). A previous study that employed the bacterial SmLPMO10A and chitin as the substrate showed a clear dependency of the LPMO reaction rate on the reductant concentration, with an apparent K M for ascorbate of 2 µM [44]. However, it is also well documented that ascorbate can reduce O 2 to H 2 O 2 under commonly employed reaction conditions [24,28].…”

Section: Ascorbate-driven Lpmo Activitymentioning

confidence: 98%

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The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

Filandr

Man

Halada

et al. 2020

Biotechnol Biofuels

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Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent redox enzymes that cleave recalcitrant biopolymers such as cellulose, chitin, starch and hemicelluloses. Although LPMOs receive ample interest in industry and academia, their reaction mechanism is not yet fully understood. Recent studies showed that H 2 O 2 is a more efficient cosubstrate for the enzyme than O 2 , which could greatly affect the utilization of LPMOs in industrial settings. Results:We probe the reactivity of LPMO9C from the cellulose-degrading fungus Neurospora crassa with a turbidimetric assay using phosphoric acid-swollen cellulose (PASC) as substrate and H 2 O 2 as a cosubstrate. The measurements were also followed by continuous electrochemical H 2 O 2 detection and LPMO reaction products were analysed by mass spectrometry. Different systems for the in situ generation of H 2 O 2 and for the reduction of LPMO's active-site copper were employed, including glucose oxidase, cellobiose dehydrogenase, and the routinely used reductant ascorbate. Conclusions:We found for all systems that the supply of H 2 O 2 limited LPMO's cellulose depolymerization activity, which supports the function of H 2 O 2 as the relevant cosubstrate. The turbidimetric assay allowed rapid determination of LPMO activity on a cellulosic substrate without the need for time-consuming and instrumentally elaborate analysis methods.

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Section: Discussionmentioning

confidence: 90%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Ascorbate-driven Lpmo Activitymentioning

confidence: 98%

See 2 more Smart Citations

The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

Filandr

Man

Halada

et al. 2020

Biotechnol Biofuels

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“…For their catalytic cycle, LPMOs require an external reductant 16 and one of two cosubstrates, molecular oxygen (O 2 ) or hydrogen peroxide (H 2 O 2 ) 17 . The cosubstrates interact with a reduced copper active site forming a reactive intermediate which can then oxidize the substrate.…”

Section: Introductionmentioning

confidence: 99%

Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

Dodge

Russo

Blossom

et al. 2020

Preprint

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Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are indispensable redox enzymes used in industry for the saccharification of plant biomass. LPMO-driven cellulose oxidation can be enhanced considerably through photobiocatalysis using chlorophyll derivatives and light. Water soluble chlorophyll binding proteins (WSCPs) make it is possible to stabilize and solubilize chlorophyll in aqueous solution, allowing for in vitro studies on photostability and ROS production. Here we aim apply a WSCP-Chl a as a photosensitizing complex for photobiocatalysis with the LPMO, TtAA9. Results We have in this study demonstrated how WSCP reconstituted with chlorophyll a (WSCP-Chl a) can create a stable photosensitizing complex which produces controlled amounts of H2O2 in the presence of ascorbic acid and light. WSCP-Chl a is highly reactive and allows for tightly controlled formation of H2O2 by regulating light intensity. TtAA9 together with WSCP-Chl a shows increased cellulose oxidation under low light conditions, and the WSCP-Chl a complex remains stable after 24 hours of light exposure. Additionally, the WSCP-Chl a complex demonstrates stability over a range of temperatures and pH conditions relevant for enzyme activity in industrial settings. Conclusion With WSCP-Chl a as the photosensitizer, the need to replenish Chl is greatly reduced, enhancing the catalytic lifetime of light-driven LPMOs and increasing the efficiency of cellulose depolymerization. WSCP-Chl a allows for stable photobiocatalysis providing a sustainable solution for biomass processing.

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Reductants fuel lytic polysaccharide monooxygenase activity in a pH‐dependent manner

et al. 2023

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Polysaccharide-degrading mono-copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H 2 O 2 cosubstrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH-dependent manner. Between pH 6.0 and 8.0, reactions with ascorbic acid show little pH dependency, whereas reactions with gallic acid become much faster at increased pH. These dependencies correlate with the reductant ionization state, which affects its ability to react with molecular oxygen and generate H 2 O 2 . The correlation does not apply to L-cysteine because, as shown by stopped-flow kinetics, increased H 2 O 2 production at higher pH is counteracted by increased binding of Lcysteine to the copper active site. The findings highlight the importance of the choice of reductant and pH in LPMO reactions.

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Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

Cited by 67 publications

References 64 publications

The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

Water-soluble chlorophyll-binding proteins from Brassica oleracea allow for stable photobiocatalytic oxidation of cellulose by a lytic polysaccharide monooxygenase

Reductants fuel lytic polysaccharide monooxygenase activity in a pH‐dependent manner

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