Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Metalloproteins perform a diverse array of redox-related reactions facilitated by the increased chemical functionality afforded by their metallocofactors. Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes that are responsible for the breakdown of recalcitrant polysaccharides via oxidative cleavage at the glycosidic bond. The activated copper-oxygen intermediates and their mechanism of formation remains to be established. Neutron protein crystallography which permits direct visualization of protonation states was used to investigate the initial steps of oxygen activation directly following active site copper reduction in Neurospora crassa LPMO9D. Herein, we cryo-trap an activated dioxygen intermediate in a mixture of superoxo and hydroperoxo states, and we identify the conserved second coordination shell residue His157 as the proton donor. Density functional theory (DFT) calculations indicate that both active site states are stable. The hydroperoxo formed is potentially an intermediate in the mechanism of hydrogen peroxide formation in the absence of substrate. We establish that the N-terminal amino group of the copper coordinating His1 remains doubly protonated directly following molecular oxygen reduction by copper. Aided by mining minima free energy calculations we establish His157 conformational flexibility in solution that is abolished by steric hindrance in the crystal. A neutron crystal structure of NcLPMO9D at low pH supports occlusion of the active site which prevents protonation of His157 at acidic conditions.

Characterisation of the enzyme transport path between shipworms and their bacterial symbionts

Background Shipworms are marine xylophagus bivalve molluscs, which can live on a diet solely of wood due to their ability to produce plant cell wall-degrading enzymes. Bacterial carbohydrate-active enzymes (CAZymes), synthesised by endosymbionts living in specialised shipworm cells called bacteriocytes and located in the animal’s gills, play an important role in wood digestion in shipworms. However, the main site of lignocellulose digestion within these wood-boring molluscs, which contains both endogenous lignocellulolytic enzymes and prokaryotic enzymes, is the caecum, and the mechanism by which bacterial enzymes reach the distant caecum lumen has remained so far mysterious. Here, we provide a characterisation of the path through which bacterial CAZymes produced in the gills of the shipworm Lyrodus pedicellatus reach the distant caecum to contribute to the digestion of wood. Results Through a combination of transcriptomics, proteomics, X-ray microtomography, electron microscopy studies and in vitro biochemical characterisation, we show that wood-digesting enzymes produced by symbiotic bacteria are localised not only in the gills, but also in the lumen of the food groove, a stream of mucus secreted by gill cells that carries food particles trapped by filter feeding to the mouth. Bacterial CAZymes are also present in the crystalline style and in the caecum of their shipworm host, suggesting a unique pathway by which enzymes involved in a symbiotic interaction are transported to their site of action. Finally, we characterise in vitro four new bacterial glycosyl hydrolases and a lytic polysaccharide monooxygenase identified in our transcriptomic and proteomic analyses as some of the major bacterial enzymes involved in this unusual biological system. Conclusion Based on our data, we propose that bacteria and their enzymes are transported from the gills along the food groove to the shipworm’s mouth and digestive tract, where they aid in wood digestion.

A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase based HRP colorimetric method for lytic polysaccharide monooxygenase activity assay

Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) are ubiquitous and diverse group of enzymes amongst the fungal kingdom. They catalyze the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for secondary biorefinery applications. Screening of AA9 LPMOs for desirable properties is crucial for biorefinery industrial applications. However, robust, high-throughput and direct method for AA9 LPMO activity assay, which is prerequisite for screening of LPMOs with excellent properties, is still lacking. Here, we have described a gluco-oligosaccharide oxidase (GOOX) based horseradish peroxidase (HRP) colorimetric method for AA9 LPMO activity assay. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and set up a SsGOOX based HRP colorimetric method for cellobiose concentration assay. Then we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G in T. reesei, purified the recombinant proteins, and analyzed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1 type (class 1) LPMO, while TtAA9G was characterized as a C4 type (class 2) LPMO. Finally, the SsGOOX based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from LPMO reaction, and consequently, the activities of both C1 and C4 types of LPMOs were analyzed. These LPMOs could be effectively analyzed with limits of detection (LoDs) lower than 30 nmol/L, and standard curves between A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1 and C4 type of AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate ready for high-throughput screening of AA9 LPMOs with high properties.