Lytic polysaccharide monooxygenases from Myceliophthora thermophila C1 differ in substrate preference and reducing agent specificity

Abstract

BackgroundLytic polysaccharide monooxgygenases (LPMOs) are known to boost the hydrolytic breakdown of lignocellulosic biomass, especially cellulose, due to their oxidative mechanism. For their activity, LPMOs require an electron donor for reducing the divalent copper cofactor. LPMO activities are mainly investigated with ascorbic acid as a reducing agent, but little is known about the effect of plant-derived reducing agents on LPMOs activity.ResultsHere, we show that three LPMOs from the fungus Myceliophthora thermophila C1, MtLPMO9A, MtLPMO9B and MtLPMO9C, differ in their substrate preference, C1-/C4-regioselectivity and reducing agent specificity. MtLPMO9A generated C1- and C4-oxidized, MtLPMO9B C1-oxidized and MtLPMO9C C4-oxidized gluco-oligosaccharides from cellulose. The recently published MtLPMO9A oxidized, next to cellulose, xylan, β-(1 → 3, 1 → 4)-glucan and xyloglucan. In addition, MtLPMO9C oxidized, to a minor extent, xyloglucan and β-(1 → 3, 1 → 4)-glucan from oat spelt at the C4 position. In total, 34 reducing agents, mainly plant-derived flavonoids and lignin-building blocks, were studied for their ability to promote LPMO activity. Reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of oxidized and non-oxidized gluco-oligosaccharides from cellulose for all three MtLPMOs. Low activities toward cellulose were observed in the presence of monophenols and sulfur-containing compounds.ConclusionsSeveral of the most powerful LPMO reducing agents of this study serve as lignin building blocks or protective flavonoids in plant biomass. Our findings support the hypothesis that LPMOs do not only vary in their C1-/C4-regioselectivity and substrate specificity, but also in their reducing agent specificity. This work strongly supports the idea that the activity of LPMOs toward lignocellulosic biomass does not only depend on the ability to degrade plant polysaccharides like cellulose, but also on their specificity toward plant-derived reducing agents in situ.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0594-y) contains supplementary material, which is available to authorized users.

Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

“…It displays activity only on polysaccharides containing (1–4)-linked glucose units (cellulose, mixed linkages beta-glucans, glucomannans and xyloglucan). However, this enzyme does not cleave plant xylans as it is the case for the AA9 LPMO from Myceliophthora thermophila which is so far the only AA9 LPMO able to cleave xylan only when complexed to cellulose29. Phosphoric acid swollen cellulose (PASC) is one of the most common substrates used to monitor cellulase activity.…”

Boosting LPMO-driven lignocellulose degradation by polyphenol oxidase-activated lignin building blocks

“…Our previous results have shown that reducing agents with a 1,2-benzenediol or 1,2,3-benzenetriol moiety gave the highest release of non-oxidized and C1-oxidized gluco-oligosaccharides from regenerated amorphous cellulose (RAC) incubated with three Mt LPMOs compared to the incubation with compounds comprising only a single hydroxyl group [13]. Hence, in the current research we hypothesized that Mt LPMO activity can benefit from enzymes which have hydroxylating capacity, such as polyphenol oxidases (Fig.…”

“…2. The 21 different phenolic compounds used were classified into three groups and further divided into subgroups, as previously described [13]. In short, group I represents compounds comprising one hydroxyl group, group II compounds comprising a 1,2-benzenediol moiety, and group III compounds comprising a 1,2,3-benzenetriol moiety (Fig.…”

“…However, monophenols are not optimal electron donors for LPMO activity, because of their relatively high redox potential [12]. Compounds with a 1,2-benzenediol or a 1,2,3-benzenetriol moiety have, compared to monophenols, a lower redox potential [13]. Their low redox potential enables them to reduce the copper ion in the active site of LPMOs and enhance the LPMO activity [12].…”

“…In addition, we used the commercially available tyrosinase Ab PPO from the white button mushroom Agaricus bisporus . Both tyrosinases were each incubated with the previously described Mt LPMO9B in the presence of various plant phenolic compounds and cellulose [13, 24]. We found that Mt PPO7 is highly active towards methoxylated monophenols, including monomeric lignin building blocks.…”