scholarly journals Lytic polysaccharide monooxygenases and other histidine-brace copper proteins: structure, oxygen activation and biotechnological applications

2021 ◽  
Author(s):  
Johan Ø. Ipsen ◽  
Magnus Hallas-Møller ◽  
Søren Brander ◽  
Leila Lo Leggio ◽  
Katja S. Johansen
Keyword(s):  
Molecular Mechanisms ◽  
Glycoside Hydrolases ◽  
Copper Proteins ◽  
Process Yield ◽  
Stirred Tank Reactors ◽  
Glycosidic Bonds ◽  
Lytic Polysaccharide Monooxygenases ◽  
Biochemical Characterisation ◽  
Polysaccharide Monooxygenases ◽  
Bacteria And Fungi

Lytic polysaccharide monooxygenases (LPMOs) are mononuclear copper enzymes that catalyse the oxidative cleavage of glycosidic bonds. They are characterised by two histidine residues that coordinate copper in a configuration termed the Cu-histidine brace. Although first identified in bacteria and fungi, LPMOs have since been found in all biological kingdoms. LPMOs are now included in commercial enzyme cocktails used in industrial biorefineries. This has led to increased process yield due to the synergistic action of LPMOs with glycoside hydrolases. However, the introduction of LPMOs makes control of the enzymatic step in industrial stirred-tank reactors more challenging, and the operational stability of the enzymes is reduced. It is clear that much is still to be learned about the interaction between LPMOs and their complex natural and industrial environments, and fundamental scientific studies are required towards this end. Several atomic-resolution structures have been solved providing detailed information on the Cu-coordination sphere and the interaction with the polysaccharide substrate. However, the molecular mechanisms of LPMOs are still the subject of intense investigation; the key question being how the proteinaceous environment controls the copper cofactor towards the activation of the O-O bond in O2 and cleavage of the glycosidic bonds in polysaccharides. The need for biochemical characterisation of each putative LPMO is discussed based on recent reports showing that not all proteins with a Cu-histidine brace are enzymes.

scholarly journals Recent Advances in Screening Methods for the Functional Investigation of Lytic Polysaccharide Monooxygenases

2021 ◽  
Vol 9 ◽  
Author(s):  
Damao Wang ◽  
Yanping Li ◽  
Yuting Zheng ◽  
Yves S. Y. Hsieh
Keyword(s):  
Copper Ions ◽  
Biomass Conversion ◽  
Screening Methods ◽  
Lytic Polysaccharide Monooxygenase ◽  
Indirect Measurements ◽  
Glycosidic Bonds ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Functional Investigation ◽  
Polysaccharide Monooxygenase

Lytic polysaccharide monooxygenase (LPMO) is a newly discovered and widely studied enzyme in recent years. These enzymes play a key role in the depolymerization of sugar-based biopolymers (including cellulose, hemicellulose, chitin and starch), and have a positive significance for biomass conversion. LPMO is a copper-dependent enzyme that can oxidize and cleave glycosidic bonds in cellulose and other polysaccharides. Their mechanism of action depends on the correct coordination of copper ions in the active site. There are still difficulties in the analysis of LPMO activity, which often requires multiple methods to be used in concert. In this review, we discussed various LPMO activity analysis methods reported so far, including mature mass spectrometry, chromatography, labeling, and indirect measurements, and summarized the advantages, disadvantages and applicability of different methods.

scholarly journals Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs)

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Riin Kont ◽  
Bastien Bissaro ◽  
Vincent G. H. Eijsink ◽  
Priit Väljamäe
Keyword(s):  
Catalytic Properties ◽  
Biomass Conversion ◽  
Side Reactions ◽  
Cellulose Oxidation ◽  
Multiple Substrates ◽  
Glycosidic Bonds ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Global Carbon ◽  
Insight Into

AbstractLytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic properties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, with kcat/KmH2O2 values in the order of 105–106 M−1 s−1. Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases.

scholarly journals Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)

2020 ◽  
pp. jbc.RA120.015545
Author(s):  
Kristian E. H. Frandsen ◽  
Mireille Haon ◽  
Sacha Grisel ◽  
Bernard Henrissat ◽  
Leila Lo Leggio ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Time Course ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Substrate Binding ◽  
Glycoside Hydrolases ◽  
Sequence Alignments ◽  
Lytic Polysaccharide Monooxygenases ◽  
Molecular Determinants ◽  
Polysaccharide Monooxygenases

Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall-derived hemicellulosic polysaccharides and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharides degradation suggests that the subtle differences in amino acids sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO sub-clusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)-linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries.

scholarly journals Lytic Polysaccharide Monooxygenases as Chitin-Specific Virulence Factors in Crayfish Plague

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1180
Author(s):  
Federico Sabbadin ◽  
Bernard Henrissat ◽  
Neil C. Bruce ◽  
Simon J. McQueen-Mason
Keyword(s):  
Endangered Species ◽  
Virulence Factors ◽  
Molecular Mechanisms ◽  
Oxidative Degradation ◽  
Aphanomyces Astaci ◽  
Lytic Polysaccharide Monooxygenases ◽  
Enzyme Family ◽  
Polysaccharide Monooxygenases ◽  
Oomycete Pathogen ◽  
Specific Virulence

The oomycete pathogen Aphanomyces astaci, also known as “crayfish plague”, is an obligate fungal-like parasite of freshwater crustaceans and is considered responsible for the ongoing decline of native European crayfish populations. A. astaci is thought to secrete a wide array of effectors and enzymes that facilitate infection, however their molecular mechanisms have been poorly characterized. Here, we report the identification of AA15 lytic polysaccharide monooxygenases (LPMOs) as a new group of secreted virulence factors in A. astaci. We show that this enzyme family has greatly expanded in A. astaci compared to all other oomycetes, and that it may facilitate infection through oxidative degradation of crystalline chitin, the most abundant polysaccharide found in the crustacean exoskeleton. These findings reveal new roles for LPMOs in animal–pathogen interactions, and could help inform future strategies for the protection of farmed and endangered species.

scholarly journals Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Claire. A. Fowler ◽  
Federico Sabbadin ◽  
Luisa Ciano ◽  
Glyn R. Hemsworth ◽  
Luisa Elias ◽  
...  
Keyword(s):  
3D Structure ◽  
Symbiotic Bacterium ◽  
Glycoside Hydrolases ◽  
Cellulosic Biomass ◽  
Cellulolytic Activity ◽  
Epr Spectrum ◽  
Saprotrophic Fungi ◽  
Lytic Polysaccharide Monooxygenases ◽  
Activity Measurements ◽  
Polysaccharide Monooxygenases

Abstract Background The quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate. Results Although classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a “histidine-brace” catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases. Conclusion Shipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.

scholarly journals Novel molecular biological tools for the efficient expression of fungal lytic polysaccharide monooxygenases in Pichia pastoris

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Lukas Rieder ◽  
Katharina Ebner ◽  
Anton Glieder ◽  
Morten Sørlie
Keyword(s):  
Pichia Pastoris ◽  
Biomass Conversion ◽  
Single Step ◽  
Additional Advantage ◽  
Lytic Polysaccharide Monooxygenases ◽  
Expression Host ◽  
Shake Flask Cultivation ◽  
Polysaccharide Monooxygenases ◽  
Efficient Expression ◽  
High Yields

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are attracting large attention due their ability to degrade recalcitrant polysaccharides in biomass conversion and to perform powerful redox chemistry. Results We have established a universal Pichia pastoris platform for the expression of fungal LPMOs using state-of-the-art recombination cloning and modern molecular biological tools to achieve high yields from shake-flask cultivation and simple tag-less single-step purification. Yields are very favorable with up to 42 mg per liter medium for four different LPMOs spanning three different families. Moreover, we report for the first time of a yeast-originating signal peptide from the dolichyl-diphosphooligosaccharide-protein glycosyltransferase subunit 1 (OST1) form S. cerevisiae efficiently secreting and successfully processes the N-terminus of LPMOs yielding in fully functional enzymes. Conclusion The work demonstrates that the industrially most relevant expression host P. pastoris can be used to express fungal LPMOs from different families in high yields and inherent purity. The presented protocols are standardized and require little equipment with an additional advantage with short cultivation periods.

scholarly journals Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Plinio S. Vieira ◽  
Isabela M. Bonfim ◽  
Evandro A. Araujo ◽  
Ricardo R. Melo ◽  
Augusto R. Lima ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Transcriptional Activation ◽  
Molecular Mechanisms ◽  
Model Organism ◽  
Citrus Canker ◽  
Effector Proteins ◽  
Glycoside Hydrolases ◽  
Host Cells ◽  
System A ◽  
Enzymatic Machinery

AbstractXyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans. Notably, the sugars released by this system elicit the expression of several key virulence factors, including the type III secretion system, a membrane-embedded apparatus to deliver effector proteins into the host cells. Together, these findings shed light on the molecular mechanisms underpinning the intricate enzymatic machinery of Xanthomonas to depolymerize xyloglucans and uncover a role for this system in signaling pathways driving pathogenesis.

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