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

Autor: Stepnov AA; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Ås, Norway., Forsberg Z; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Ås, Norway., Sørlie M; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Ås, Norway., Nguyen GS; Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway., Wentzel A; Department of Biotechnology and Nanomedicine, SINTEF Industry, Trondheim, Norway., Røhr ÅK; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Ås, Norway., Eijsink VGH; Faculty of Chemistry, Biotechnology and Food Science, NMBU-Norwegian University of Life Sciences, Ås, Norway. vincent.eijsink@nmbu.no.
Jazyk: angličtina
Zdroj: Biotechnology for biofuels [Biotechnol Biofuels] 2021 Jan 21; Vol. 14 (1), pp. 28. Date of Electronic Publication: 2021 Jan 21.
DOI: 10.1186/s13068-021-01879-0
Abstrakt: 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 O 2 or H 2 O 2 . 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 H 2 O 2 , 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 H 2 O 2 resulting from oxidation of the reductant.
Conclusion: The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O 2 , 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 H 2 O 2 , the (O 2 -driven) LPMO reaction is exceedingly slow, compared to the much faster peroxygenase reaction that occurs when adding H 2 O 2 .
Databáze: MEDLINE
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