Abstract
Background: Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative
depolymerization of industrially relevant crystalline polysaccharides, such as cellulose, in a reaction that depends on
an electron donor and O2 or H2O2. While it is well known that LPMOs can utilize a wide variety of electron donors, the
variation in reported efciencies of various LPMO-reductant combinations remains largely unexplained.
Results: In this study, we describe a novel two-domain cellulose-active family AA10 LPMO from a marine actinomy‑
cete, which we have used to look more closely at the efects of the reductant and copper ions on the LPMO reaction.
Our results show that ascorbate-driven LPMO reactions are extremely sensitive to very low amounts (micromolar
concentrations) of free copper because reduction of free Cu(II) ions by ascorbic acid leads to formation of H2O2, which
speeds up the LPMO reaction. In contrast, the use of gallic acid yields steady reactions that are almost insensitive to
the presence of free copper ions. Various experiments, including dose–response studies with the enzyme, showed
that under typically used reaction conditions, the rate of the reaction is limited by LPMO-independent formation of
H2O2 resulting from oxidation of the reductant.
Conclusion: The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O2, i.e. the
most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates.
The observed diferences between ascorbic acid and gallic acid show a way of making LPMO reactions less copperdependent and illustrate that reductant efects on LPMO action need to be interpreted with great caution. In clean
reactions, with minimized generation of H2O2, the (O2-driven) LPMO reaction is exceedingly slow, compared to the
much faster peroxygenase reaction that occurs when adding H2O2.
depolymerization of industrially relevant crystalline polysaccharides, such as cellulose, in a reaction that depends on
an electron donor and O2 or H2O2. While it is well known that LPMOs can utilize a wide variety of electron donors, the
variation in reported efciencies of various LPMO-reductant combinations remains largely unexplained.
Results: In this study, we describe a novel two-domain cellulose-active family AA10 LPMO from a marine actinomy‑
cete, which we have used to look more closely at the efects of the reductant and copper ions on the LPMO reaction.
Our results show that ascorbate-driven LPMO reactions are extremely sensitive to very low amounts (micromolar
concentrations) of free copper because reduction of free Cu(II) ions by ascorbic acid leads to formation of H2O2, which
speeds up the LPMO reaction. In contrast, the use of gallic acid yields steady reactions that are almost insensitive to
the presence of free copper ions. Various experiments, including dose–response studies with the enzyme, showed
that under typically used reaction conditions, the rate of the reaction is limited by LPMO-independent formation of
H2O2 resulting from oxidation of the reductant.
Conclusion: The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O2, i.e. the
most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates.
The observed diferences between ascorbic acid and gallic acid show a way of making LPMO reactions less copperdependent and illustrate that reductant efects on LPMO action need to be interpreted with great caution. In clean
reactions, with minimized generation of H2O2, the (O2-driven) LPMO reaction is exceedingly slow, compared to the
much faster peroxygenase reaction that occurs when adding H2O2.