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Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

Sørensen, H. V. LU ; Montserrat-Canals, Mateu ; Loose, Jennifer S.M. ; Fisher, S. Zoë LU ; Moulin, Martine ; Blakeley, Matthew P. ; Cordara, Gabriele ; Bjerregaard-Andersen, Kaare and Krengel, Ute (2023) In ACS Omega 8(32). p.29101-29112
Abstract

Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and... (More)

Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields >10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.

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organization
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type
Contribution to journal
publication status
published
subject
in
ACS Omega
volume
8
issue
32
pages
12 pages
publisher
The American Chemical Society (ACS)
external identifiers
  • scopus:85167930910
  • pmid:37599915
ISSN
2470-1343
DOI
10.1021/acsomega.3c02168
language
English
LU publication?
yes
id
62c9e6f3-e089-4384-8afe-e5a63d436381
date added to LUP
2023-10-30 14:47:19
date last changed
2024-04-05 00:48:19
@article{62c9e6f3-e089-4384-8afe-e5a63d436381,
  abstract     = {{<p>Lytic polysaccharide monooxygenases (LPMOs) are surface-active redox enzymes that catalyze the degradation of recalcitrant polysaccharides, making them important tools for energy production from renewable sources. In addition, LPMOs are important virulence factors for fungi, bacteria, and viruses. However, many knowledge gaps still exist regarding their catalytic mechanism and interaction with their insoluble, crystalline substrates. Moreover, conventional structural biology techniques, such as X-ray crystallography, usually do not reveal the protonation state of catalytically important residues. In contrast, neutron crystallography is highly suited to obtain this information, albeit with significant sample volume requirements and challenges associated with hydrogen’s large incoherent scattering signal. We set out to demonstrate the feasibility of neutron-based techniques for LPMOs using N-acetylglucosamine-binding protein A (GbpA) from Vibrio cholerae as a target. GbpA is a multifunctional protein that is secreted by the bacteria to colonize and degrade chitin. We developed an efficient deuteration protocol, which yields &gt;10 mg of pure 97% deuterated protein per liter expression media, which was scaled up further at international facilities. The deuterated protein retains its catalytic activity and structure, as demonstrated by small-angle X-ray and neutron scattering studies of full-length GbpA and X-ray crystal structures of its LPMO domain (to 1.1 Å resolution), setting the stage for neutron scattering experiments with its substrate chitin.</p>}},
  author       = {{Sørensen, H. V. and Montserrat-Canals, Mateu and Loose, Jennifer S.M. and Fisher, S. Zoë and Moulin, Martine and Blakeley, Matthew P. and Cordara, Gabriele and Bjerregaard-Andersen, Kaare and Krengel, Ute}},
  issn         = {{2470-1343}},
  language     = {{eng}},
  number       = {{32}},
  pages        = {{29101--29112}},
  publisher    = {{The American Chemical Society (ACS)}},
  series       = {{ACS Omega}},
  title        = {{Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase}},
  url          = {{http://dx.doi.org/10.1021/acsomega.3c02168}},
  doi          = {{10.1021/acsomega.3c02168}},
  volume       = {{8}},
  year         = {{2023}},
}