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Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase.

ABSTRACT: Lytic polysaccharide monooxygenases (LPMOs) are copper-containing metalloenzymes that can cleave the glycosidic link in polysaccharides. This could become crucial for production of energy-efficient biofuels from recalcitrant polysaccharides. Although LPMOs are considered oxygenases, recent investigations have shown that H2O2 can also act as a co-substrate for LPMOs. Intriguingly, LPMOs generate H2O2 in the absence of a polysaccharide substrate. Here, we elucidate a new mechanism for H2O2 generation starting from an AA10-LPMO crystal structure with an oxygen species bound, using QM/MM calculations. The reduction level and protonation state of this oxygen-bound intermediate has been unclear. However, this information is crucial to the mechanism. We therefore investigate the oxygen-bound intermediate with quantum refinement (crystallographic refinement enhanced with QM calculations), against both X-ray and neutron data. Quantum refinement calculations suggest a Cu(ii)-O-2 system in the active site of the AA10-LPMO and a neutral protonated -NH2 state for the terminal nitrogen atom, the latter in contrast to the original interpretation. Our QM/MM calculations show that H2O2 generation is possible only from a Cu(i) center and that the most favourable reaction pathway is to involve a nearby glutamate residue, adding two electrons and two protons to the Cu(ii)-O-2 system, followed by dissociation of H2O2.

SUBMITTER: Caldararu O

PROVIDER: S-EPMC6334667 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase.

Caldararu Octav O Oksanen Esko E Ryde Ulf U Hedegård Erik D ED

Chemical science 20181019 2

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing metalloenzymes that can cleave the glycosidic link in polysaccharides. This could become crucial for production of energy-efficient biofuels from recalcitrant polysaccharides. Although LPMOs are considered oxygenases, recent investigations have shown that H2O2 can also act as a co-substrate for LPMOs. Intriguingly, LPMOs generate H2O2 in the absence of a polysaccharide substrate. Here, w ...[more]

PMID: 30746099

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Project description:Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that perform oxidative cleavage of recalcitrant polysaccharides. We have purified and characterized a recombinant family AA9 LPMO, LPMO9B, from Gloeophyllum trabeum (GtLPMO9B) which is active on both cellulose and xyloglucan. Activity of the enzyme was tested in the presence of three different reductants: ascorbic acid, gallic acid, and 2,3-dihydroxybenzoic acid (2,3-DHBA). Under standard aerobic conditions typically used in LPMO experiments, the first two reductants could drive LPMO catalysis whereas 2,3-DHBA could not. In agreement with the recent discovery that H2O2 can drive LPMO catalysis, we show that gradual addition of H2O2 allowed LPMO activity at very low, substoichiometric (relative to products formed) reductant concentrations. Most importantly, we found that while 2,3-DHBA is not capable of driving the LPMO reaction under standard aerobic conditions, it can do so in the presence of externally added H2O2 At alkaline pH, 2,3-DHBA is able to drive the LPMO reaction without externally added H2O2, and this ability overlaps entirely the endogenous generation of H2O2 by GtLPMO9B-catalyzed oxidation of 2,3-DHBA. These findings support the notion that H2O2 is a cosubstrate of LPMOs and provide insight into how LPMO reactions depend on, and may be controlled by, the choice of pH and reductant.IMPORTANCE Lytic polysaccharide monooxygenases promote enzymatic depolymerization of lignocellulosic materials by microorganisms due to their ability to oxidatively cleave recalcitrant polysaccharides. The properties of these copper-dependent enzymes are currently of high scientific and industrial interest. We describe a previously uncharacterized fungal LPMO and show how reductants, which are needed to prime the LPMO by reducing Cu(II) to Cu(I) and to supply electrons during catalysis, affect enzyme efficiency and stability. The results support claims that H2O2 is a natural cosubstrate for LPMOs by demonstrating that when certain reductants are used, catalysis can be driven only by H2O2 and not by O2 Furthermore, we show how auto-inactivation resulting from endogenous generation of H2O2 in the LPMO-reductant system may be prevented. Finally, we identified a reductant that leads to enzyme activation without any endogenous H2O2 generation, allowing for improved control of LPMO reactivity and providing a valuable tool for future LPMO research.

Dataset Information

Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase.

Publications

Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase.

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