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 Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

2018 ◽  
Vol 294 (5) ◽  
pp. 1516-1528 ◽  
Author(s):  
Silja Kuusk ◽  
Riin Kont ◽  
Piret Kuusk ◽  
Agnes Heering ◽  
Morten Sørlie ◽  
...  
Keyword(s):  
Reduction Reaction ◽  
Reaction Conditions ◽  
Lytic Polysaccharide Monooxygenase ◽  
Glycosidic Bonds ◽  
Initial Reduction ◽  
Catalytically Active ◽  
Polysaccharide Monooxygenases ◽  
Theoretical Analyses ◽  
Polysaccharide Monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of 14C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of 14C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O2. When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a “priming reduction” reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (KmRapp). Theoretical analyses revealed that KmRapp decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O2, H2O2, and polysaccharide concentrations.

scholarly journals Lytic polysaccharide monooxygenase (LPMO) mediated production of ultra-fine cellulose nanofibres from delignified softwood fibres

2019 ◽  
Vol 21 (21) ◽  
pp. 5924-5933 ◽  
Author(s):  
Salla Koskela ◽  
Shennan Wang ◽  
Dingfeng Xu ◽  
Xuan Yang ◽  
Kai Li ◽  
...  
Keyword(s):  
Efficient Method ◽  
Energy Efficient ◽  
Environmentally Friendly ◽  
Oxidative Enzymes ◽  
Lytic Polysaccharide Monooxygenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Cellulose Nanofibres ◽  
Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenase

An environmentally friendly, energy-efficient method for cellulose nanofibre (CNF) production from softwood holocellulose utilising oxidative enzymes, lytic polysaccharide monooxygenases (LPMOs).

scholarly journals The role of the active site tyrosine in the mechanism of lytic polysaccharide monooxygenase

2021 ◽  
Vol 12 (1) ◽  
pp. 352-362
Author(s):  
Aina McEvoy ◽  
Joel Creutzberg ◽  
Raushan K. Singh ◽  
Morten J. Bjerrum ◽  
Erik D. Hedegård
Keyword(s):  
Active Site ◽  
Lytic Polysaccharide Monooxygenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Active Site Tyrosine ◽  
Polysaccharide Monooxygenase

With QM/MM, we investigate the mechanism of tyrosine deprotonation in lytic polysaccharide monooxygenases. Our results support deprotonation and our calculated UV-vis spectra show that two isomers must be formed to match recent experiments.

scholarly journals Mechanism of hydrogen peroxide formation by lytic polysaccharide monooxygenase

2019 ◽  
Vol 10 (2) ◽  
pp. 576-586 ◽  
Author(s):  
Octav Caldararu ◽  
Esko Oksanen ◽  
Ulf Ryde ◽  
Erik D. Hedegård
Keyword(s):  
Hydrogen Peroxide ◽  
Peroxide Formation ◽  
Lytic Polysaccharide Monooxygenase ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Hydrogen Peroxide Formation ◽  
Polysaccharide Monooxygenase

A mechanism for the formation of hydrogen peroxide by lytic polysaccharide monooxygenases (LPMOs) in the absence of substrate is proposed.

Combination of MALDI-TOF MS and UHPLC-ESI-MS for the characterization of lytic polysaccharide monooxygenase activity

2020 ◽  
Vol 12 (2) ◽  
pp. 149-161 ◽  
Author(s):  
Caio de Oliveira Gorgulho Silva ◽  
Tallyta Santos Teixeira ◽  
Kelly Barreto Rodrigues ◽  
Amanda Araújo Souza ◽  
Antonielle Vieira Monclaro ◽  
...  
Keyword(s):  
Lytic Polysaccharide Monooxygenase ◽  
Monooxygenase Activity ◽  
Maldi Tof Ms ◽  
Esi Ms ◽  
Maldi Tof ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Tof Ms ◽  
Polysaccharide Monooxygenase

Two new mass spectrometry methods, MALDI-TOF MS and hydrophilic interaction UHPLC-ESI-MS, were developed for the characterization of cellulose-active lytic polysaccharide monooxygenases, expanding the analytical toolbox for the study of these enzymes.

scholarly journals Further structural studies of the lytic polysaccharide monooxygenase AoAA13 belonging to the starch-active AA13 family

Amylase ◽  
2019 ◽  
Vol 3 (1) ◽  
pp. 41-54 ◽  
Author(s):  
Sebastian J. Muderspach ◽  
Tobias Tandrup ◽  
Kristian E. H. Frandsen ◽  
Gianluca Santoni ◽  
Jens-Christian N. Poulsen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Metal Binding ◽  
Binding Studies ◽  
Orthorhombic Crystal ◽  
Lytic Polysaccharide Monooxygenase ◽  
X Ray ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Triclinic Structure ◽  
Polysaccharide Monooxygenase

Abstract Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper enzymes that cleave recalcitrant polysaccharides by oxidation. The structure of an Aspergillus oryzae LPMO from the starch degrading family AA13 (AoAA13) has previously been determined from an orthorhombic crystal grown in the presence of copper, which is photoreduced in the structure. Here we describe how crystals reliably grown in presence of Zn can be Cu-loaded post crystallization. A partly photoreduced structure was obtained by severely limiting the X-ray dose, showing that this LPMO is much more prone to photoreduction than others. A serial synchrotron crystallography structure was also obtained, showing that this technique may be promising for further studies, to reduce even further photoreduction. We additionally present a triclinic structure of AoAA13, which has less occluded ligand binding site than the orthorhombic one. The availability of the triclinic crystals prompted new ligand binding studies, which lead us to the conclusion that small starch analogues do not bind to AoAA13 to an appreciable extent. A number of disordered conformations of the metal binding histidine brace have been encountered in this and other studies, and we have previously hypothesized that this disorder may be a consequence of loss of copper. We performed molecular dynamics in the absence of active site metal, and showed that the dynamics in solution differ somewhat from the disorder observed in the crystal, though the extent is equally dramatic.

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 Molecular mechanism of the chitinolytic peroxygenase reaction

2020 ◽  
Vol 117 (3) ◽  
pp. 1504-1513 ◽  
Author(s):  
Bastien Bissaro ◽  
Bennett Streit ◽  
Ingvild Isaksen ◽  
Vincent G. H. Eijsink ◽  
Gregg T. Beckham ◽  
...  
Keyword(s):  
Defense Mechanisms ◽  
Biomass Conversion ◽  
Copper Catalysts ◽  
Fast Process ◽  
Lytic Polysaccharide Monooxygenases ◽  
Crystalline Substrate ◽  
In Silico Studies ◽  
Initial Cleavage ◽  
Polysaccharide Monooxygenases ◽  
Network Of Hydrogen Bonds

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of monocopper enzymes broadly distributed across the tree of life. Recent reports indicate that LPMOs can use H2O2 as an oxidant and thus carry out a novel type of peroxygenase reaction involving unprecedented copper chemistry. Here, we present a combined computational and experimental analysis of the H2O2-mediated reaction mechanism. In silico studies, based on a model of the enzyme in complex with a crystalline substrate, suggest that a network of hydrogen bonds, involving both the enzyme and the substrate, brings H2O2 into a strained reactive conformation and guides a derived hydroxyl radical toward formation of a copper–oxyl intermediate. The initial cleavage of H2O2 and subsequent hydrogen atom abstraction from chitin by the copper–oxyl intermediate are the main energy barriers. Stopped-flow fluorimetry experiments demonstrated that the priming reduction of LPMO–Cu(II) to LPMO–Cu(I) is a fast process compared to the reoxidation reactions. Using conditions resulting in single oxidative events, we found that reoxidation of LPMO–Cu(I) is 2,000-fold faster with H2O2 than with O2, the latter being several orders of magnitude slower than rates reported for other monooxygenases. The presence of substrate accelerated reoxidation by H2O2, whereas reoxidation by O2 became slower, supporting the peroxygenase paradigm. These insights into the peroxygenase nature of LPMOs will aid in the development and application of enzymatic and synthetic copper catalysts and contribute to a further understanding of the roles of LPMOs in nature, varying from biomass conversion to chitinolytic pathogenesis-defense mechanisms.

Lytic Polysaccharide Monooxygenases in Biomass Conversion

2015 ◽  
Vol 33 (12) ◽  
pp. 747-761 ◽  
Author(s):  
Glyn R. Hemsworth ◽  
Esther M. Johnston ◽  
Gideon J. Davies ◽  
Paul H. Walton
Keyword(s):  
Biomass Conversion ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases
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