Abstract
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 kcat value of 124 ± 27 s–1 at 4 °C is 20 times higher than a previously described kcat 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.
Highlights
Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative scission of glycosidic bonds and by doing so they boost the activity of classical polysaccharide-degrading hydrolytic enzymes such as chitinases and cellulases.[1−10] LPMO catalytic sites contain a single copper-ion cofactor[11,12] that upon reduction reacts with either O2 or H2O2 to generate oxygen species that is capable of abstracting a hydrogen atom from the C1 or the C4 carbon atom in glycosidic bonds.[9,13−16]
Rapid enzyme inactivation under peroxygenase conditions may be taken to indicate that the peroxygenase reaction is not a true LPMO reaction[21] but could have other explanations, such as subsaturating substrate concentrations that leave the enzyme prone to damaging off pathway reactions with H2O2.16,20,22 Importantly, under the conditions typically used in LPMO “monooxygenase” reactions, H2O2 will be generated in situ and Hydroxylation of one of the carbons destabilizes the glycosidic bond, which, once oxidized, undergoes an elimination reaction leading to bond breakage.[12]
The experiments described above show two important aspects of LPMO enzymology. They illustrate that it is complicated to properly assess LPMO catalysis experimentally, due to the plethora of interconnected reactions. Many of these complications emerged in our experiments and by studying multiple reductants, each with its own peculiarities, we were able to overcome most of these complications and generate insights into LPMO catalysis
Summary
Enzymes currently known as lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative scission of glycosidic bonds and by doing so they boost the activity of classical polysaccharide-degrading hydrolytic enzymes such as chitinases and cellulases.[1−10] LPMO catalytic sites contain a single copper-ion cofactor[11,12] that upon reduction reacts with either O2 or H2O2 to generate oxygen species that is capable of abstracting a hydrogen atom from the C1 or the C4 carbon atom in glycosidic bonds.[9,13−16]Initially, LPMOs were thought to be monooxygenases[3] (Figure 1A), but recent studies have shown that LPMOs can act as peroxygenases[15] (Figure 1B) and that this reaction is faster than the monooxygenase reaction.[15−20] The peroxygenase reaction tends to lead to more enzyme damage compared to the monooxygenase reaction and may lead to reduced catalytic specificity.[21]. Stepnov et al.[24] showed that the generation of H2O2 in standard reactions with an AA10 type (bacterial) LPMO (i.e., LPMO + 1 mM reductant) was almost independent of the LPMO in reactions with gallic acid (GA), whereas the LPMO increased H2O2 production in reactions with ascorbic acid (AscA). It is not known whether the same would apply for the AA9 LPMOs that are abundant in biomass-degrading fungi
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