Fast and Specific Peroxygenase Reactions Catalyzed by Fungal Mono-Copper Enzymes

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 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.

Peroxide-Mediated Oxygenation of Organic Compounds by Fungal Peroxygenases

“…For the latter, a molecular architecture would be required that allows the superficial targeting of aromatic polymer structures via heme access channels that are as broad and shallow as possible. In this context, it should be noted that, according to recent publications, the crystalline cellulose-cleaving LPMOs (lytic polysaccharide monooxygenases), which were discovered only a few years ago, are also surface-acting extracellular peroxygenases (albeit based on copper-dependent catalysis) [ 125 , 137 , 138 ].…”

Concluding remarks: discussion on natural and artificial enzymes including synthetic models

“…7). [35][36][37][38][39][40][41][42] The active site has a single copper ion with three protein derived ligands, a terminal histidine forming a chelate via coordination of the amino terminus and an imidazole nitrogen atom (the "histidine brace"), along with a second histidine derived imidazole donor (Fig. 7).…”

Characterization of a Dual Cellulolytic/Xylanolytic AA9 Lytic Polysaccharide Monooxygenase from Thermothelomyces thermophilus and its Utilization Toward Nanocellulose Production in a Multi-Step Bioprocess

“…Being able to utilize both O 2 and H 2 O 2 as a co-substrate and many different compounds as electron donors, their activity as monooxygenases and peroxygenases has been debated. 15,16 They may also participate in non-specific Fenton-type reactions due to their copper-containing active site. 17 Many different LPMO genes have been found in the genome of a variety of lignocellulose-degrading microorganisms, thus raising questions about their functional properties.…”