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Discovery and characterization of a new family of lytic polysaccharide monooxygenases.

Hemsworth GR, Henrissat B, Davies GJ, Walton PH - Nat. Chem. Biol. (2013)

Bottom Line: They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels.Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33).The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of York, Heslington, York, UK.

ABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.

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Structural comparisons of Ao(AA11) with known AA9 and AA10 enzymes. (a) 3D structure of Cu-Ao(AA11), ribbon depiction. The conserved active site residues are shown as sticks with green carbons and disulfide bonds (from conserved cysteines) as yellow sticks. (b) Overall superposition of Cu-Ao(AA11) (green) with Zn-(AA9) from T. terrestris (yellow) with rmsd = 2.6 Å over 145 Cα’s (c) Superposition of Cu-Ao(AA11) (green) with Cu-(AA10) from E. faecaelis (pink) with r.m.s.d = 2.3 Å over 118 residues overlapping with a Cα’s. (d) The electron density maps contoured at 1σ in the active site of Cu-Ao(AA11), Cu-N(His 1) = 1.97 Å, Cu-NH2(His1) = 2.19 Å, Cu-N(His60) = 1.98 Å, N(His1)-Cu-NH2 = 90.5°, N(His60)-Cu-NH2 = 103.0°, N(His1)-Cu-N(His1) = 164.8°. Glu74, marked with asterisk is from a symmetry related molecule and is shown with yellow carbon atoms. (e) Active site overlay of Ao(AA11) (green carbons/copper) with Cu-AA9 from T. aurantiacus (orange carbons/copper), note side chain of conserved alanine 58, depicted as green rod in AA11. (f) The active site overlap of Cu-Ao(AA11) (green carbons/copper) with Cu-(AA10) from B. amyloliquefaciens (pink carbons/copper). See Supplementary figure 5 for stereo views of d-f.
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Figure 4: Structural comparisons of Ao(AA11) with known AA9 and AA10 enzymes. (a) 3D structure of Cu-Ao(AA11), ribbon depiction. The conserved active site residues are shown as sticks with green carbons and disulfide bonds (from conserved cysteines) as yellow sticks. (b) Overall superposition of Cu-Ao(AA11) (green) with Zn-(AA9) from T. terrestris (yellow) with rmsd = 2.6 Å over 145 Cα’s (c) Superposition of Cu-Ao(AA11) (green) with Cu-(AA10) from E. faecaelis (pink) with r.m.s.d = 2.3 Å over 118 residues overlapping with a Cα’s. (d) The electron density maps contoured at 1σ in the active site of Cu-Ao(AA11), Cu-N(His 1) = 1.97 Å, Cu-NH2(His1) = 2.19 Å, Cu-N(His60) = 1.98 Å, N(His1)-Cu-NH2 = 90.5°, N(His60)-Cu-NH2 = 103.0°, N(His1)-Cu-N(His1) = 164.8°. Glu74, marked with asterisk is from a symmetry related molecule and is shown with yellow carbon atoms. (e) Active site overlay of Ao(AA11) (green carbons/copper) with Cu-AA9 from T. aurantiacus (orange carbons/copper), note side chain of conserved alanine 58, depicted as green rod in AA11. (f) The active site overlap of Cu-Ao(AA11) (green carbons/copper) with Cu-(AA10) from B. amyloliquefaciens (pink carbons/copper). See Supplementary figure 5 for stereo views of d-f.

Mentions: Screening for crystallization conditions for apo-Ao(AA11) identified a condition containing 10 mM ZnCl2 (see methods) which gave diffraction quality crystals. The resulting structure was solved by single wavelength anomalous dispersion at λ = 1.282 Å, optimising the f” component of the zinc anomalous scattering. The Zn-Ao(AA11) structure was refined to a final resolution of 1.55 Å (Supplementary Table 1) with a single Zn2+ ion in the active site. The necessity for Zn2+ in the crystallisation condition was revealed by the direct coordination to the zinc ion by the side chain of Glu74 from a symmetry-related molecule, thereby augmenting the packing interaction between adjacent AA11 molecules. The structure of Cu-Ao(AA11), Fig. 4a, was obtained via soaking of the Zn-crystal in cryo-protectant containing 2 mM Cu2+. Consistent with the ITC described above and the expected relative binding constants of zinc and copper from the Irving-Williams series, this allowed access to the Cu-bound form of AoAA11. Structural analysis of Cu-Ao(AA11) at 1.4 Å (Supplementary Table 1) shows that it has a similar tertiary structure to the AA9 and AA10 classes of LPMOs. The core of the protein is formed by a largely antiparallel β-sandwich fold and is stabilised by three disulfide bonds. In both Zn-Ao(AA11) and Cu-Ao(AA11) structures, residues 99–109 and 151–169 are highly disordered and have not been modelled in the final structure. The intact nature of the enzyme was confirmed however by electrospray mass spectrometry of apo-Ao(AA11). Both of these mobile regions are adjacent to the C-terminal region of the structure where the AA11 domain was truncated to remove the X278 module. It is possible that these regions will be ordered in the intact multi-domain protein.


Discovery and characterization of a new family of lytic polysaccharide monooxygenases.

Hemsworth GR, Henrissat B, Davies GJ, Walton PH - Nat. Chem. Biol. (2013)

Structural comparisons of Ao(AA11) with known AA9 and AA10 enzymes. (a) 3D structure of Cu-Ao(AA11), ribbon depiction. The conserved active site residues are shown as sticks with green carbons and disulfide bonds (from conserved cysteines) as yellow sticks. (b) Overall superposition of Cu-Ao(AA11) (green) with Zn-(AA9) from T. terrestris (yellow) with rmsd = 2.6 Å over 145 Cα’s (c) Superposition of Cu-Ao(AA11) (green) with Cu-(AA10) from E. faecaelis (pink) with r.m.s.d = 2.3 Å over 118 residues overlapping with a Cα’s. (d) The electron density maps contoured at 1σ in the active site of Cu-Ao(AA11), Cu-N(His 1) = 1.97 Å, Cu-NH2(His1) = 2.19 Å, Cu-N(His60) = 1.98 Å, N(His1)-Cu-NH2 = 90.5°, N(His60)-Cu-NH2 = 103.0°, N(His1)-Cu-N(His1) = 164.8°. Glu74, marked with asterisk is from a symmetry related molecule and is shown with yellow carbon atoms. (e) Active site overlay of Ao(AA11) (green carbons/copper) with Cu-AA9 from T. aurantiacus (orange carbons/copper), note side chain of conserved alanine 58, depicted as green rod in AA11. (f) The active site overlap of Cu-Ao(AA11) (green carbons/copper) with Cu-(AA10) from B. amyloliquefaciens (pink carbons/copper). See Supplementary figure 5 for stereo views of d-f.
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Figure 4: Structural comparisons of Ao(AA11) with known AA9 and AA10 enzymes. (a) 3D structure of Cu-Ao(AA11), ribbon depiction. The conserved active site residues are shown as sticks with green carbons and disulfide bonds (from conserved cysteines) as yellow sticks. (b) Overall superposition of Cu-Ao(AA11) (green) with Zn-(AA9) from T. terrestris (yellow) with rmsd = 2.6 Å over 145 Cα’s (c) Superposition of Cu-Ao(AA11) (green) with Cu-(AA10) from E. faecaelis (pink) with r.m.s.d = 2.3 Å over 118 residues overlapping with a Cα’s. (d) The electron density maps contoured at 1σ in the active site of Cu-Ao(AA11), Cu-N(His 1) = 1.97 Å, Cu-NH2(His1) = 2.19 Å, Cu-N(His60) = 1.98 Å, N(His1)-Cu-NH2 = 90.5°, N(His60)-Cu-NH2 = 103.0°, N(His1)-Cu-N(His1) = 164.8°. Glu74, marked with asterisk is from a symmetry related molecule and is shown with yellow carbon atoms. (e) Active site overlay of Ao(AA11) (green carbons/copper) with Cu-AA9 from T. aurantiacus (orange carbons/copper), note side chain of conserved alanine 58, depicted as green rod in AA11. (f) The active site overlap of Cu-Ao(AA11) (green carbons/copper) with Cu-(AA10) from B. amyloliquefaciens (pink carbons/copper). See Supplementary figure 5 for stereo views of d-f.
Mentions: Screening for crystallization conditions for apo-Ao(AA11) identified a condition containing 10 mM ZnCl2 (see methods) which gave diffraction quality crystals. The resulting structure was solved by single wavelength anomalous dispersion at λ = 1.282 Å, optimising the f” component of the zinc anomalous scattering. The Zn-Ao(AA11) structure was refined to a final resolution of 1.55 Å (Supplementary Table 1) with a single Zn2+ ion in the active site. The necessity for Zn2+ in the crystallisation condition was revealed by the direct coordination to the zinc ion by the side chain of Glu74 from a symmetry-related molecule, thereby augmenting the packing interaction between adjacent AA11 molecules. The structure of Cu-Ao(AA11), Fig. 4a, was obtained via soaking of the Zn-crystal in cryo-protectant containing 2 mM Cu2+. Consistent with the ITC described above and the expected relative binding constants of zinc and copper from the Irving-Williams series, this allowed access to the Cu-bound form of AoAA11. Structural analysis of Cu-Ao(AA11) at 1.4 Å (Supplementary Table 1) shows that it has a similar tertiary structure to the AA9 and AA10 classes of LPMOs. The core of the protein is formed by a largely antiparallel β-sandwich fold and is stabilised by three disulfide bonds. In both Zn-Ao(AA11) and Cu-Ao(AA11) structures, residues 99–109 and 151–169 are highly disordered and have not been modelled in the final structure. The intact nature of the enzyme was confirmed however by electrospray mass spectrometry of apo-Ao(AA11). Both of these mobile regions are adjacent to the C-terminal region of the structure where the AA11 domain was truncated to remove the X278 module. It is possible that these regions will be ordered in the intact multi-domain protein.

Bottom Line: They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels.Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33).The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of York, Heslington, York, UK.

ABSTRACT
Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.

Show MeSH
Related in: MedlinePlus