Trichoderma harzianum Peptaibols Stimulate Plant Plasma Membrane H+-ATPase Activity.

Because of their ability to promote growth, act as biopesticides, and improve abiotic stress tolerance, Trichoderma spp. have been used for plant seed coating. However, the mechanism for the promotion of plant growth remains unknown. In this study, we investigate the effect of fungal extracts on the plant plasma membrane (PM) H+-ATPase, which is essential for plant growth and often a target of plant-associated microbes. We show that Trichoderma harzianum extract increases H+-ATPase activity, and by fractionation and high-resolution mass spectrometry (MS), we identify the activating components trichorzin PA (tPA) II and tPA VI that belong to the class of peptaibols. Peptaibols are nonribosomal peptides that can integrate into membranes and form indiscriminate ion channels, which causes pesticidal activity. To further investigate peptaibol-mediated H+-ATPase activation, we compare the effect of tPA II and VI to that of the model peptaibol alamethicin (AlaM). We show that AlaM increases H+-ATPase turnover rates in a concentration-dependent manner, with a peak in activity measured at 31.25 µM, above which activity decreases. Using fluorescent probes and light scattering, we find that the AlaM-mediated increase in activity is not correlated to increased membrane fluidity or vesicle integrity, whereas the activity decrease at high AlaM concentrations is likely due to PM overloading of AlaM pores. Overall, our results suggest that the symbiosis of fungi and plants, specifically related to peptaibols, is a concentration-dependent balance, where peptaibols do not act only as biocontrol agents but also as plant growth stimulants.

ATP hydrolytic activity of purified Spf1p correlate with micellar lipid fluidity and is dependent on conserved residues in transmembrane helix M1.

P5A ATPases are expressed in the endoplasmic reticulum (ER) of all eukaryotic cells, and their disruption results in pleiotropic phenotypes related to severe ER stress. They were recently proposed to function in peptide translocation although their specificity have yet to be confirmed in reconstituted assays using the purified enzyme. A general theme for P-type ATPases is that binding and transport of substrates is coupled to hydrolysis of ATP in a conserved allosteric mechanism, however several independent reports have shown purified Spf1p to display intrinsic spontaneous ATP hydrolytic activity after purification. It has never been determined to what extend this spontaneous activity is caused by uncoupling of the enzyme. In this work we have purified a functional tagged version of the Saccharomyces cerevisiae P5A ATPase Spf1p and have observed that the intrinsic ATP hydrolytic activity of the purified and re-lipidated protein can be stimulated by specific detergents (C12E8, C12E10 and Tween20) in mixed lipid/detergent micelles in the absence of any apparent substrate. We further show that this increase in activity correlate with the reaction temperature and the anisotropic state of the mixed lipid/detergent micelles and further that this correlation relies on three highly conserved phenylalanine residues in M1. This suggests that at least part of the intrinsic ATP hydrolytic activity is allosterically coupled to movements in the TM domain in the purified preparations. It is suggested that free movement of the M1 helix represent an energetic constraint on catalysis and that this constraint likely is lost in the purified preparations resulting in protein with intrinsic spontaneous ATP hydrolytic activity. Removal of the N-terminal part of the protein apparently removes this activity.

Protonation State of an Important Histidine from High Resolution Structures of Lytic Polysaccharide Monooxygenases.

Lytic Polysaccharide Monooxygenases (LPMOs) oxidatively cleave recalcitrant polysaccharides. The mechanism involves (i) reduction of the Cu, (ii) polysaccharide binding, (iii) binding of different oxygen species, and (iv) glycosidic bond cleavage. However, the complete mechanism is poorly understood and may vary across different families and even within the same family. Here, we have investigated the protonation state of a secondary co-ordination sphere histidine, conserved across AA9 family LPMOs that has previously been proposed to be a potential proton donor. Partial unrestrained refinement of newly obtained higher resolution data for two AA9 LPMOs and re-refinement of four additional data sets deposited in the PDB were carried out, where the His was refined without restraints, followed by measurements of the His ring geometrical parameters. This allowed reliable assignment of the protonation state, as also validated by following the same procedure for the His brace, for which the protonation state is predictable. The study shows that this histidine is generally singly protonated at the Nε2 atom, which is close to the oxygen species binding site. Our results indicate robustness of the method. In view of this and other emerging evidence, a role as proton donor during catalysis is unlikely for this His.

Inhibition of LPMOs by Fermented Persimmon Juice.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.

Inhibition of lytic polysaccharide monooxygenase by natural plant extracts.

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 activity-modulating 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.

The synergy between LPMOs and cellulases in enzymatic saccharification of cellulose is both enzyme- and substrate-dependent.

OBJECTIVES: The synergistic effects between cellulases and lytic polysaccharide monooxygenases (LPMOs) were investigated systematically in terms of their degree of synergy (DS) on amorphous and crystalline cellulose. Synergy curves were obtained for enzyme pairs containing a cellulase from Trichoderma reesei (Cel6A and Cel7A) and three LPMOs from Thermoascus aurantiacus (TaAA9A), Lentinus similis (LsAA9A) and Thielavia terrestris (TtAA9E). RESULTS: The synergistic experiments showed that the three LPMOs significantly improved the hydrolytic efficiency of Cel6A, on both cellulosic substrates; a more pronounced effect being seen for TtAA9E on amorphous cellulose at low cellulase:LPMO ratios. In contrast, the highly processive, reducing-end acting Cel7A synergised with the C1-C4 oxidising LPMOs, TaAA9A and LsAA9A, but was inhibited by the presence of C1-oxidizing TtAA9E. CONCLUSIONS: The degree of synergy exhibited by the cellulase-LPMO mixtures was enzyme- and substrate-specific. The observed Cel7A inhibition, rather than synergy, by the C1-oxidizing LPMO, TtAA9E, warrants further investigations.