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

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 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 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.

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.

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.

scholarly journals Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

Biomolecules ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1098
Author(s):  
Federica Calderaro ◽  
Loes E. Bevers ◽  
Marco A. van den Berg
Keyword(s):  
Experimental Design ◽  
Mode Of Action ◽  
Data Interpretation ◽  
Electron Donors ◽  
Cellulolytic Enzymes ◽  
Inhibiting Factors ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Power Tools ◽  
Reliable Methods

Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cellulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples.

Protein expression and extraction of hard-to-produce proteins in the periplasmic space of Escherichia coli v1

2021 ◽  
Author(s):  
Cristina Hernandez Rollan ◽  
Kristoffer Bach Falkenberg ◽  
Maja Rennig ◽  
Andreas Birk Bertelsen ◽  
Morten Norholm
Keyword(s):  
Protein Production ◽  
Expression System ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
E Coli ◽  
New Family ◽  
Lytic Polysaccharide Monooxygenases ◽  
Strain Bl21 ◽  
Polysaccharide Monooxygenases

E. coli is a gram-negative bacteria used mainly in academia and in some industrial scenarios, as a protein production workhorse. This is due to its ease of manipulation and the range of genetic tools available. This protocol describes how to express proteins in the periplasm E. coli with the strain BL21 (DE3) using a T7 expression system. Specifically, it describes a series of steps and tips to express "hard-to-express" proteins in E. coli, as for instance, LPMOs. The protocol is adapted from Hemsworth, G. R., Henrissat, B., Davies, G. J., and Walton, P. H. (2014) Discovery and characterization of a new family of lytic polysaccharide monooxygenases. Nat. Chem. Biol.10, 122–126. .

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