Unraveling the roles of the reductant and free copper ions in LPMO kinetics

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 efficiencies 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 actinomycete, which we have used to look more closely at the effects 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 differences between ascorbic acid and gallic acid show a way of making LPMO reactions less copper-dependent and illustrate that reductant effects 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.

Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity

“…This shows that indeed LPMO is limiting the apparent monooxygenase reaction, which is in contrast to observations made for other LPMOs whose reactions may be limited by H 2 O 2 -generating LPMO-independent side reactions involving the reductant and O 2 . 53 Importantly, the rate at the intercept (0 μM LPMO) was significant (0.0184 μM*s –1 ). Of note, this “LPMO-independent” background level of substrate conversion is effectively inhibited by HRP ( Figure 3 A,B), which shows that this conversion involves H 2 O 2 generated in solution, likely resulting from auto-oxidation of ascorbic acid 53 and is not due to, for example, a true monooxygenase reaction that would not involve H 2 O 2 .…”

Combining pieces: a thorough analysis of light activation boosting power and co-substrate preferences for the catalytic efficiency of lytic polysaccharide monooxygenase MtLPMO9A

“…2b). (Stepnov et al, 2021). Furthermore, molecular dynamic simulations using the quantum mechanics/molecular mechanics (QM/MM) method have demonstrated that ascorbate rapidly reduces LPMO-Cu(II) in a thermodynamically favorable process (Wang et al, 2019;Wang et al, 2020).…”

Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

“…Another aspect to consider when using this assay is the amount of H 2 O 2 generated by the reaction of the reductant with oxygen. Stepnov et al compared the effects of different amounts of gallic or ascorbic acid, highlighting the striking effect that free copper in LPMO preparations can have on the determination of LPMO rates [78]. with the Amplex TM Red assay before (green and blue lines) and after (brown and red lines) enzyme purification.…”