Scission of Glucosidic Bonds by a Lentinus similis Lytic Polysaccharide Monooxygenases Is Strictly Dependent on H₂O₂ while the Oxidation of Saccharide Products Depends on O₂

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that act in synergy with glycoside hydrolases to saccharify the most abundant polysaccharides in nature. Both O 2 and H 2 O 2 can be cosubstrates for LPMOs. The Lentinus similis LPMO (LsAA9A) has previously been shown to oxidatively cleave oligosaccharides when supplied with ascorbate as a reductant. This study demonstrates that LsAA9A is unable to complete the catalytic cycle and cleave cellulose without H 2 O 2 . Instead, cellooligomers efficiently prevent the slow continuous oxidation of ascorbate taking place under ambient conditions in the absence of a substrate. LsAA9A specifically cleaves cellooligomers in a fast and stoichiometric reaction with H 2 O 2 as a cosubstrate. However, the product profile contains more non-oxidized saccharides than anticipated by the generally accepted LPMO reaction scheme. The scission of glucosidic bonds and oxidation of the saccharide therefore appear not to be directly coupled. This was confirmed by the complete absence of oxidized products under anoxic conditions. A mechanism is proposed involving the hydrolysis of a cellulosic radical formed by a H 2 O 2 -derived caged hydroxyl radical. In addition, LsAA9A catalyzes another stoichiometric reaction with excess H 2 O 2 to oxidize ascorbate in the absence of cellulose. Ascorbate is not a cosubstrate for the reaction leading to the scission of glucosidic bonds by LsAA9A.

Changes in active-site geometry on X-ray photoreduction of a lytic polysaccharide monooxygenase active-site copper and saccharide binding

“…Both O 2 and H 2 O 2 have been suggested as natural co-substrates (Bissaro et al, 2017; and the LPMO mechanism is being assessed continuously for the involvement of either/both (Bissaro et al, 2020;Hedega ˚rd & Ryde, 2017;Courtade et al, 2020;McEvoy et al, 2021;Brander et al, 2020). The dependence on H 2 O 2 for saccharide cleavage has been established recently specifically for one of the model LPMOs investigated here (Brander, Tokin et al, 2021), and is generally gaining increasing support. The H 2 O 2 -driven mechanism has the advantage that the LPMO active-site metal only needs to be reduced once, while in the O 2 -driven mechanism, it needs to be reduced at every cycle (Bissaro et al, 2017).…”

“…In vitro reductants such as ascorbic acid, gallic acid or cysteine are commonly used (Wang et al, 2020). As well as being essential for further reactivity, there are several reports indicating increased polysaccharide affinity on reduction of the active-site metal, suggesting that polysaccharide binding precedes further steps in the mechanism (Kracher et al, 2018;Filandr et al, 2020;Brander, Tokin et al, 2021). Both O 2 and H 2 O 2 have been suggested as natural co-substrates (Bissaro et al, 2017; and the LPMO mechanism is being assessed continuously for the involvement of either/both (Bissaro et al, 2020;Hedega ˚rd & Ryde, 2017;Courtade et al, 2020;McEvoy et al, 2021;Brander et al, 2020).…”

Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases

“…Computational studies on Ls AA9A have also shown that the H 2 O 2 co-substrate does not require proton transfer (and thus double protonation) [ 27 , 33 ]. A new experimental study further highlights that H 2 O 2 is the co-substrate strictly required for Ls AA9A glycosidic bond cleavage activity [ 28 ]. Thus, at least for Ls AA9A there is currently no evidence of His147 double protonation or proton transfer being necessary for the mechanism.…”

“…The black arrow in Figure 1 (pointing to the chloride ion) denotes the position of the oxygen-species binding site. The oxygen species could be provided by either molecular O 2 or H 2 O 2 , although the support for the latter is increasing [ 25 , 26 , 27 , 28 ]. After binding, the oxygen species is positioned appropriately near the substrate by the residues around the active site.…”

“…This is likely followed by hydrogen abstraction from the substrate, hydroxylation and elimination of water, resulting in oxidation at either C1 or C4 atoms. However, recent detailed work on Ls AA9A shows that while glycosidic bond cleavage is dependent on H 2 O 2 , O 2 is necessary for the production of oxidated oligosaccharides [ 28 ]. This suggests that rather than a strict preference for either co-substrates, at least some LPMOs follow more complicated reaction schemes where interactions between metal, oxygen species, protein and reductant determine the final outcome, which is highly sensitive to the reaction conditions.…”