Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Schröder, Gabriela C.; O’Dell, William B.; Webb, S. P.; Agarwal, Pratul K.; Meilleur, Flora

doi:10.1039/d2sc05031e

Cited by 21 publications

(26 citation statements)

References 110 publications

(195 reference statements)

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“…6b). This confirms that in this conformation His147 remains neutral at low pH (Schro ¨der et al, 2022), as also shown by analysis of high-resolution X-ray structures (Banerjee et al, 2022).…”

Section: Residues Near the Active Site: Tyr164 And His147supporting

confidence: 86%

“…Neutron protein crystallography is a powerful tool for investigating protein chemistry because it directly locates H-atom positions in a protein structure (Schro ¨der et al, 2018). Early X-ray and neutron crystallographic studies of LPMOs from the AA9 family have focused on understanding the activation of O 2 by Neurospora crassa LPMO 9D (NcAA9_D) in the absence of substrate (Bodenheimer et al, 2017;O'Dell, Swartz et al, 2017;O'Dell, Agarwal et al, 2017;Schro ¨der et al, , 2022. In addition to the direct determination of protonation states, these structures have revealed the geometry and the chemical nature of the initial Cu-O 2 À and Cu-O 2 H intermediates.…”

Section: Introductionmentioning

confidence: 99%

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Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature

Tandrup

Leggio

Meilleur

2023

Acta Crystallogr F Struct Biol Commun

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Lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes which cleave polysaccharides oxidatively and are important in pathogen biology, carbon cycling and biotechnology. The Lentinus similis family AA9 isoform A (LsAA9_A) has been extensively studied as a model system because its activity towards smaller soluble saccharide substrates has allowed detailed structural characterization of its interaction with a variety of substrates by X-ray crystallography at high resolution. Here, the joint X-ray/neutron room-temperature crystallographic structure of carbohydrate-free LsAA9_A in the copper(II) resting state refined against X-ray and neutron data at 2.1 and 2.8 Å resolution, respectively, is presented. The results provide an experimental determination of the protonation states of the copper(II)-coordinating residues and second-shell residues in LsAA9_A, paving the way for future neutron crystallographic studies of LPMO–carbohydrate complexes.

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Section: Residues Near the Active Site: Tyr164 And His147supporting

confidence: 86%

Section: Introductionmentioning

confidence: 99%

Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature

Tandrup

Leggio

Meilleur

2023

Acta Crystallogr F Struct Biol Commun

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“…The H 2 O 2 molecule was placed such as to interact with His155 and Gln/Glu164 based on the results of previous computational studies. ,,, These previous studies have shown that LPMO reactivity is associated with an intricate fluctuating H-bonding network, which in the current model depends on the protonation state of His155 (HID or HIE tautomeric forms or HIP) and Glu164 (for the mutant). Recent neutron diffraction and high-resolution X-ray diffraction studies on other AA9s have shown His155 likely does not occur in the double protonated form, not even at acidic pH. , The initial reactivity calculations along the HO–OH bond cleavage pathway featured His155 with the proton at the N ε position (HIE). The choice of HIE155 is supported by neutron diffraction studies , of other AA9s from Neurospora crassa and the higher stability of HIE compared to HID in QM/MM calculations .…”

Section: Resultsmentioning

confidence: 99%

“…Recent neutron diffraction and high-resolution X-ray diffraction studies on other AA9s have shown His155 likely does not occur in the double protonated form, not even at acidic pH. , The initial reactivity calculations along the HO–OH bond cleavage pathway featured His155 with the proton at the N ε position (HIE). The choice of HIE155 is supported by neutron diffraction studies , of other AA9s from Neurospora crassa and the higher stability of HIE compared to HID in QM/MM calculations . The lack of similar data on the Q164E mutant motivated us to explore both protonation states of Glu164.…”

Section: Resultsmentioning

confidence: 99%

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases

Hall,

Joseph,

Ayuso-Fernández

et al. 2023

J. Am. Chem. Soc.

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Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C–H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H2O2 splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.

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“…This alleviates the sample volume requirement by as much as a factor of ten. A few neutron crystal structures of LPMOs have already been determined; − however, none of them is perdeuterated, enabling mapping of only select hydrogen atoms.…”

Section: Introductionmentioning

confidence: 99%

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

Sørensen,

Montserrat-Canals,

Loose

et al. 2023

ACS Omega

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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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Capture of activated dioxygen intermediates at the copper-active site of a lytic polysaccharide monooxygenase

Abstract: Superoxo and hydroperoxo intermediates were cryotrapped at the copper active site of lytic polysaccharide monooxygenase using neutron protein crystallography.

Cited by 21 publications

References 110 publications

Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature

Joint X-ray/neutron structure of Lentinus similis AA9_A at room temperature

A Conserved Second Sphere Residue Tunes Copper Site Reactivity in Lytic Polysaccharide Monooxygenases

Perdeuterated GbpA Enables Neutron Scattering Experiments of a Lytic Polysaccharide Monooxygenase

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