scholarly journals Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina

2015 ◽  
Vol 8 (1) ◽  
Author(s):  
Chloé Bennati-Granier ◽  
Sona Garajova ◽  
Charlotte Champion ◽  
Sacha Grisel ◽  
Mireille Haon ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Podospora Anserina ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

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 Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases

2014 ◽  
Vol 7 (1) ◽  
Author(s):  
Adam J Book ◽  
Ragothaman M Yennamalli ◽  
Taichi E Takasuka ◽  
Cameron R Currie ◽  
George N Phillips ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

scholarly journals Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

2012 ◽  
Vol 79 (2) ◽  
pp. 488-496 ◽  
Author(s):  
Mathieu Bey ◽  
Simeng Zhou ◽  
Laetitia Poidevin ◽  
Bernard Henrissat ◽  
Pedro M. Coutinho ◽  
...  
Keyword(s):  
Cellulose Hydrolysis ◽  
Podospora Anserina ◽  
Striking Difference ◽  
Glycoside Hydrolase Family ◽  
Carbohydrate Binding ◽  
Pycnoporus Cinnabarinus ◽  
Content Type ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

ABSTRACTThe genome of the coprophilic ascomycetePodospora anserinaencodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserinaGH61A [PaGH61A] andPaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced inPichia pastoris. Synergistic cooperation betweenPaGH61A orPaGH61B with the cellobiose dehydrogenase (CDH) ofPycnoporus cinnabarinuson cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference betweenPaGH61A andPaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination ofPaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties ofPaGH61A andPaGH61B and their effect on the interaction with CDH are discussed in regard to the proposedin vivofunction of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

scholarly journals Sequence and Structural Analysis of AA9 and AA10 LPMOs: An Insight into the Basis of Substrate Specificity and Regioselectivity

2019 ◽  
Vol 20 (18) ◽  
pp. 4594 ◽  
Author(s):  
Xiaoli Zhou ◽  
Xiaohua Qi ◽  
Hongxia Huang ◽  
Honghui Zhu
Keyword(s):  
Structural Analysis ◽  
Substrate Specificity ◽  
Molecular Basis ◽  
Substrate Binding ◽  
Binding Modes ◽  
Substrate Specificities ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Natural Carbon ◽  
Insight Into

Lytic polysaccharide monooxygenases (LPMOs) are key enzymes in both the natural carbon cycle and the biorefinery industry. Understanding the molecular basis of LPMOs acting on polysaccharide substrates is helpful for improving industrial cellulase cocktails. Here we analyzed the sequences, structures, and substrate binding modes of LPMOs to uncover the factors that influence substrate specificity and regioselectivity. Our results showed that the different compositions of a motif located on L2 affect the electrostatic potentials of substrate binding surfaces, which in turn affect substrate specificities of AA10 LPMOs. A conserved Asn at a distance of 7 Å from the active center Cu might, together with the conserved Ser immediately before the second catalytic His, determine the localization of LPMOs on substrate, and thus contribute to C4-oxidizing regioselectivity. The findings in this work provide an insight into the molecular basis of substrate specificity and regioselectivity of LPMOs.

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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