The effect of linker conformation on performance and stability of a two-domain lytic polysaccharide monooxygenase

publishedVersio

Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field

A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates (phosphoric acid swollen cellulose, konjac glucomannan) and has an ability to hydrolyse xylan that is lacking in TfCel9A.publishedVersio

Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field

A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates (phosphoric acid swollen cellulose, konjac glucomannan) and has an ability to hydrolyse xylan that is lacking in TfCel9A

Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field

A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates (phosphoric acid swollen cellulose, konjac glucomannan) and has an ability to hydrolyse xylan that is lacking in TfCel9A

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

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 O2 or H2O2. While it is well known that LPMOs can utilize a wide variety of electron donors, the variation in reported efciencies 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 actinomy‑ cete, which we have used to look more closely at the efects 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 H2O2, 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 H2O2 resulting from oxidation of the reductant. Conclusion: The strong impact of low amounts of free copper on LPMO reactions with ascorbic acid and O2, i.e. the most commonly used conditions when assessing LPMO activity, likely explains reported variations in LPMO rates. The observed diferences between ascorbic acid and gallic acid show a way of making LPMO reactions less copperdependent and illustrate that reductant efects on LPMO action need to be interpreted with great caution. In clean reactions, with minimized generation of H2O2, the (O2-driven) LPMO reaction is exceedingly slow, compared to the much faster peroxygenase reaction that occurs when adding H2O

DetIE: Multilingual Open Information Extraction Inspired by Object Detection

State of the art neural methods for open information extraction (OpenIE) usually extract triplets (or tuples) iteratively in an autoregressive or predicate-based manner in order not to produce duplicates. In this work, we propose a different approach to the problem that can be equally or more successful. Namely, we present a novel single-pass method for OpenIE inspired by object detection algorithms from computer vision. We use an order-agnostic loss based on bipartite matching that forces unique predictions and a Transformer-based encoder-only architecture for sequence labeling. The proposed approach is faster and shows superior or similar performance in comparison with state of the art models on standard benchmarks in terms of both quality metrics and inference time. Our model sets the new state of the art performance of 67.7% F1 on CaRB evaluated as OIE2016 while being 3.35x faster at inference than previous state of the art. We also evaluate the multilingual version of our model in the zero-shot setting for two languages and introduce a strategy for generating synthetic multilingual data to fine-tune the model for each specific language. In this setting, we show performance improvement of 15% on multilingual Re-OIE2016, reaching 75% F1 for both Portuguese and Spanish languages. Code and models are available at https://github.com/sberbank-ai/DetIE

Limited proteolysis of E. coli ATP-dependent protease Lon - a unified view of the subunit architecture and characterization of isolated enzyme fragments

We carried out chymotryptic digestion of multimeric ATP-dependent Lon protease from Escherichia coli. Four regions sensitive to proteolytic digestion were located in the enzyme and several fragments corresponding to the individual structural domains of the enzyme or their combinations were isolated. It was shown that (i) unlike the known AAA+ proteins, the ATPase fragment (A) of Lon has no ATPase activity in spite of its ability to bind nucleotides, and it is monomeric in solution regardless of the presence of any effectors; (ii) the monomeric proteolytic domain (P) does not display proteolytic activity; (iii) in contrast to the inactive counterparts, the AP fragment is an oligomer and exhibits both the ATPase and proteolytic activities. However, unlike the full-length Lon, its AP fragment oligomerizes into a dimer or a tetramer only, exhibits the properties of a non-processive protease, and undergoes self-degradation upon ATP hydrolysis. These results reveal the crucial role played by the non-catalytic N fragment of Lon (including its coiled-coil region), as well as the contribution of individual domains to creation of the quaternary structure of the full-length enzyme, empowering its function as a processive protease

Structural and functional characterization of the catalytic domain of a cell-wall anchored bacterial lytic polysaccharide monooxygenase from Streptomyces coelicolor

Abstract Bacterial lytic polysaccharide monooxygenases (LPMOs) are known to oxidize the most abundant and recalcitrant polymers in Nature, namely cellulose and chitin. The genome of the model actinomycete Streptomyces coelicolor A3(2) encodes seven putative LPMOs, of which, upon phylogenetic analysis, four group with typical chitin-oxidizing LPMOs, two with typical cellulose-active LPMOs, and one which stands out by being part of a subclade of non-characterized enzymes. The latter enzyme, called ScLPMO10D, and most of the enzymes found in this subclade are unique, not only because of variation in the catalytic domain, but also as their C-terminus contains a cell wall sorting signal (CWSS), which flags the LPMO for covalent anchoring to the cell wall. Here, we have produced a truncated version of ScLPMO10D without the CWSS and determined its crystal structure, EPR spectrum, and various functional properties. While showing several structural and functional features typical for bacterial cellulose active LPMOs, ScLPMO10D is only active on chitin. Comparison with two known chitin-oxidizing LPMOs of different taxa revealed interesting functional differences related to copper reactivity. This study contributes to our understanding of the biological roles of LPMOs and provides a foundation for structural and functional comparison of phylogenetically distant LPMOs with similar substrate specificities