Inhibition of lytic polysaccharide monooxygenase by natural plant extracts

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes of industrial and biological importance. In particular, LPMOs play important roles in fungal lifestyle. No inhibitors of LPMOs have yet been reported.In this study, a diverse library of 100 plant extracts was screened for LPMO activitymodulating effects. By employing protein crystallography and LC-MS, we successfully identified a natural LPMO inhibitor.Extract screening revealed a significant LPMO inhibition by methanolic extract of Cinnamomum cassia (cinnamon), which inhibited LsAA9A LPMO from Lentinus similis in a concentration-dependent manner. With a notable exception, other microbial LPMOs from families AA9 and AA10 were also inhibited by this cinnamon extract. The polyphenol cinnamtannin B1 was identified as the inhibitory component by crystallography. Cinnamtannin B1 was bound to the surface of LsAA9A at two distinct binding sites: one close to the active site and another at a pocket on the opposite side of the protein. Independent characterization of cinnamon extract by LC-MS and subsequent activity measurements confirmed that the compound inhibiting LsAA9A was cinnamtannin B1.The results of this study show that specific natural LPMO inhibitors of plant origin exist in nature, providing the opportunity for future exploitation of such compounds within various biotechnological contexts.

Inhibition of LPMOs by Fermented Persimmon Juice

“…Once again, alleviation of LPMO inhibition was successfully achieved with the addition of PEG. In a recent study, a polyphenolic inhibitor from an extract of cinnamon, cinnamtannin B1, was shown to bind to the surface of an LPMO [ 9 ]. In this study, TaAA9A was reported to have lower susceptibility to inhibition by the cinnamon extract during PASC degradation, in contrast to TfLPMO10A, which showed the highest.…”

“…This co-substrate did not affect TaAA9A activity and inhibition under the tested conditions ( Figure A1 ), which is an indication that Kakishibu inhibition did not stem from potential H 2 O 2 -scavenging properties of the juice. A number of small phenolic compounds were detected in Kakishibu juice ( Table A1 ), among which is the well-known LPMO reductant gallic acid, as well as catechin, taxifolin, and quercetin, which have also been reported as LPMO activators [ 9 ]. The positive effect of complex biomass mixtures has been reported previously, such as TaAA9A activation by pretreated corn stover [ 24 ], as well as activation by pretreated wheat straw for T. reesei and S. marcescens LPMOs [ 46 ].…”

“…The positive effect of complex biomass mixtures has been reported previously, such as TaAA9A activation by pretreated corn stover [ 24 ], as well as activation by pretreated wheat straw for T. reesei and S. marcescens LPMOs [ 46 ]. Furthermore, a recent study reported both inhibition and activation of another cellulose-active LPMO from L. similis by a number of methanolic and aqueous plant extracts [ 9 ].…”

“…The polyphenolic fingerprint of Kakishibu was analyzed using liquid chromatography–mass spectrometry (LC-ESI-MS/MS, Thermo Fisher Scientific, Waltham, MA, USA), as described previously [ 9 ]. In brief, Kakishibu was filtered through a 0.22 μm membrane filter, in order to remove aggregated polyphenols, and injected into a Zorbax Eclipse Plus C-18 column (1.8 μm particle size, Agilent Technologies, Glostrup, Denmark) fitted to a UHPLC system, combined with an Orbitrap Q Exactive high-resolution mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA).…”

On the expansion of biological functions of lytic polysaccharide monooxygenases

“…Also, this study makes us wonder about the evolutionary relationship, if any, between LPMO‐derived oxidised oligosaccharides and plant cellular receptors (Liu et al ., 2012; Shinya et al ., 2015). Interestingly, we underscore that the secretion of chemicals by the plant could also be used as a means to regulate fungal LPMO activity, as a very recent report has shown that some plant extracts (containing cinnamtannin B1) acted as a potent LPMO inhibitor (Tokin et al ., 2021).…”