scholarly journals Insights from semi-oriented EPR spectroscopy studies into the interaction of lytic polysaccharide monooxygenases with cellulose

2020 ◽  
Vol 49 (11) ◽  
pp. 3413-3422 ◽  
Author(s):  
Luisa Ciano ◽  
Alessandro Paradisi ◽  
Glyn R. Hemsworth ◽  
Morten Tovborg ◽  
Gideon J. Davies ◽  
...  
Keyword(s):  
Epr Spectroscopy ◽  
Active Site ◽  
Crystalline Cellulose ◽  
Cellulose Fibril ◽  
Cellulose Substrate ◽  
Lytic Polysaccharide Monooxygenases ◽  
Natural Cellulose ◽  
Polysaccharide Monooxygenases ◽  
Spectroscopy Studies

Semi-orientated EPR spectroscopy reveals that lytic polysaccharide monooxygenases interact with their natural cellulose substrate in a specific way, where the copper active site is positioned adjacent to the edge of a crystalline cellulose fibril.

Recent insights into lytic polysaccharide monooxygenases (LPMOs)

2018 ◽  
Vol 46 (6) ◽  
pp. 1431-1447 ◽  
Author(s):  
Tobias Tandrup ◽  
Kristian E. H. Frandsen ◽  
Katja S. Johansen ◽  
Jean-Guy Berrin ◽  
Leila Lo Leggio
Keyword(s):  
Active Site ◽  
Room Temperature ◽  
Experimental Studies ◽  
Binding Mode ◽  
Structural Features ◽  
Oxygen Species ◽  
Animal Kingdom ◽  
X Ray ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper enzymes discovered within the last 10 years. By degrading recalcitrant substrates oxidatively, these enzymes are major contributors to the recycling of carbon in nature and are being used in the biorefinery industry. Recently, two new families of LPMOs have been defined and structurally characterized, AA14 and AA15, sharing many of previously found structural features. However, unlike most LPMOs to date, AA14 degrades xylan in the context of complex substrates, while AA15 is particularly interesting because they expand the presence of LPMOs from the predominantly microbial to the animal kingdom. The first two neutron crystallography structures have been determined, which, together with high-resolution room temperature X-ray structures, have putatively identified oxygen species at or near the active site of LPMOs. Many recent computational and experimental studies have also investigated the mechanism of action and substrate-binding mode of LPMOs. Perhaps, the most significant recent advance is the increasing structural and biochemical evidence, suggesting that LPMOs follow different mechanistic pathways with different substrates, co-substrates and reductants, by behaving as monooxygenases or peroxygenases with molecular oxygen or hydrogen peroxide as a co-substrate, respectively.

scholarly journals Formation of a Copper(II)–Tyrosyl Complex at the Active Site of Lytic Polysaccharide Monooxygenases Following Oxidation by H2O2

2019 ◽  
Vol 141 (46) ◽  
pp. 18585-18599 ◽  
Author(s):  
Alessandro Paradisi ◽  
Esther M. Johnston ◽  
Morten Tovborg ◽  
Callum R. Nicoll ◽  
Luisa Ciano ◽  
...  
Keyword(s):  
Active Site ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

scholarly journals In vitro biosynthesis of poly-β-1,4-glucan derivatives using a pro-miscuous glycosyltransferase

2020 ◽  
Author(s):  
Gregory S. Bulmer ◽  
Ashley P. Mattey ◽  
Fabio Parmeggiani ◽  
Ryan Williams ◽  
Helene Ledru ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Chemical Probes ◽  
Cell Wall Degrading Enzymes ◽  
Lytic Polysaccharide Monooxygenases ◽  
Natural Cellulose ◽  
Polysaccharide Monooxygenases ◽  
In Vitro Biosynthesis ◽  
New Applications ◽  
Biosynthetic Machinery

AbstractThe β-1,4-glucose linkage of cellulose is the most abundant polymeric linkage on earth and as such is of considerable interest in biology and biotechnology. It remains challenging to synthesize this linkage in vitro due to a lack of suitable biocatalysts; the natural cellulose biosynthetic machinery is a membrane-associated complex with processive activity that cannot be easily manipulated to synthesize tailor-made oligosaccharides and their derivatives. Here we identify a promiscuous activity of a soluble recombinant biocatalyst, Neisseria meningitidis glycosyltransferase LgtB, suitable for the polymerization of glucose from UDP-glucose via the generation of β-1,4-glycosidic linkages. We employed LgtB to synthesize natural and derivatized cello-oligosaccharides and we demonstrate how LgtB can be incorporated in biocatalytic cascades and chemo-enzymatic strategies to synthesize cello-oligosaccharides with tailored functionalities. We also show how the resulting glycan structures can be applied as chemical probes to report on activity and selectivity of plant cell wall degrading enzymes, including lytic polysaccharide monooxygenases. We anticipate that this biocatalytic approach to derivatized cello-oligosaccharides via glucose polymerization will open up new applications in biology and nanobiotechnology.

scholarly journals Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Peicheng Sun ◽  
Christophe V. F. P. Laurent ◽  
Stefan Scheiblbrandner ◽  
Matthias Frommhagen ◽  
Dimitrios Kouzounis ◽  
...  
Keyword(s):  
Active Site ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

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 Structural Dynamics of Lytic Polysaccharide Monooxygenase during Catalysis

Biomolecules ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 242
Author(s):  
Frantisek Filandr ◽  
Daniel Kavan ◽  
Daniel Kracher ◽  
Christophe V.F.P. Laurent ◽  
Roland Ludwig ◽  
...  
Keyword(s):  
Active Site ◽  
Structural Changes ◽  
Side Chain ◽  
Crystalline Cellulose ◽  
Structural Determinants ◽  
Lytic Polysaccharide Monooxygenase ◽  
Time Resolved ◽  
Polysaccharide Monooxygenases ◽  
Hydrogen Deuterium ◽  
Partial Unfolding

Lytic polysaccharide monooxygenases (LPMOs) are industrially important oxidoreductases employed in lignocellulose saccharification. Using advanced time-resolved mass spectrometric techniques, we elucidated the structural determinants for substrate-mediated stabilization of the fungal LPMO9C from Neurospora crassa during catalysis. LPMOs require a reduction in the active-site copper for catalytic activity. We show that copper reduction in NcLPMO9C leads to structural rearrangements and compaction around the active site. However, longer exposure to the reducing agent ascorbic acid also initiated an uncoupling reaction of the bound oxygen species, leading to oxidative damage, partial unfolding, and even fragmentation of NcLPMO9C. Interestingly, no changes in the hydrogen/deuterium exchange rate were detected upon incubation of oxidized or reduced LPMO with crystalline cellulose, indicating that the LPMO-substrate interactions are mainly side-chain mediated and neither affect intraprotein hydrogen bonding nor induce significant shielding of the protein surface. On the other hand, we observed a protective effect of the substrate, which slowed down the autooxidative damage induced by the uncoupling reaction. These observations further complement the picture of structural changes during LPMO catalysis.

Modelling a ‘histidine brace’ motif in mononuclear copper monooxygenases

2020 ◽  
Vol 56 (38) ◽  
pp. 5123-5126
Author(s):  
Arisa Fukatsu ◽  
Yuma Morimoto ◽  
Hideki Sugimoto ◽  
Shinobu Itoh
Keyword(s):  
Copper Complex ◽  
Active Site ◽  
Methane Monooxygenase ◽  
Particulate Methane Monooxygenase ◽  
Site Model ◽  
Lytic Polysaccharide Monooxygenases ◽  
Active Site Model ◽  
Polysaccharide Monooxygenases

A mononuclear copper complex bearing a ‘histidine brace’ is synthesised and characterised as an active-site model of mononuclear copper monooxygenases such as lytic polysaccharide monooxygenases (LPMOs) and particulate methane monooxygenase (pMMO).

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.

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