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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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The X-band EPR spectra of Cu enzymes (2500-3500 G, 9.3 GHz, 150 K) with simulations (red) of a) Cu-Ao(AA11), pH 5, 10% v/v glycerol and b) Cu-Ao(A11), pH 5, 10% v/v glycerol with 1000 equivalents of azide.
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Figure 5: The X-band EPR spectra of Cu enzymes (2500-3500 G, 9.3 GHz, 150 K) with simulations (red) of a) Cu-Ao(AA11), pH 5, 10% v/v glycerol and b) Cu-Ao(A11), pH 5, 10% v/v glycerol with 1000 equivalents of azide.

Mentions: The X-band EPR spectra of Cu-Ao(AA11) at 150 K in the absence and presence of azide are shown in Fig. 5. The azide-free structure exhibits a near-axial spectral envelope revealing a singly-occupied molecular orbital at copper with significant d(x2-y2) character. Accurate simulation of the Cu-Ao(AA11) spectrum could be achieved in the parallel region with gz = 2.27 and ∣Az∣ = 157 G (0.0166 cm−1, Supplementary Table 2), placing Cu-Ao(AA11) squarely within the Peisach-Blumberg classification of a type 2 copper centre.25 Given the lack of resolution in the perpendicular region, accurate simulation of the Ax,y and of gx,y values was not possible, however an overall fit to the spectral envelope could only be achieved by introducing a degree of rhombicity, with gx = 2.03 and gy = 2.10. It is notable that there is some resolution of superhyperfine coupling to ligating nitrogen atoms observable in the perpendicular region (~15 G, 0.0014 cm−1, 43 MHz) with coupling constants typical for coordination to copper(II) by sp2 hybridised nitrogen atoms.26 Addition of excess sodium azide causes a shift in the EPR spectrum of Cu-Ao(AA11) to gx = gy = 2.06, gz = 2.24 and ∣Az∣ = 175 G (0.018 cm−1, Fig. 5, Supplementary Table 2) demonstrating that azide coordinates directly to the copper ion, accompanied by a shift in coordination geometry symmetry towards axial. A comparison of the known EPR parameters for the different LPMO classes and substrates, using the standard axial type 2 copper parameters in AA9 as a reference (Supplementary Table 3) shows that AA10 enzymes which are active on chitin demonstrate a reduced ∣Az∣ value and some rhombicity in perpendicular g values. AA11 enzymes have type 2 copper ∣Az∣ values and rhombicity in g values, thereby lying somewhat in-between AA9 and AA10 in terms of their EPR spectroscopic features, commensurate with the structural variations in the active sites of the different classes.


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

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

The X-band EPR spectra of Cu enzymes (2500-3500 G, 9.3 GHz, 150 K) with simulations (red) of a) Cu-Ao(AA11), pH 5, 10% v/v glycerol and b) Cu-Ao(A11), pH 5, 10% v/v glycerol with 1000 equivalents of azide.
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Related In: Results  -  Collection

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Figure 5: The X-band EPR spectra of Cu enzymes (2500-3500 G, 9.3 GHz, 150 K) with simulations (red) of a) Cu-Ao(AA11), pH 5, 10% v/v glycerol and b) Cu-Ao(A11), pH 5, 10% v/v glycerol with 1000 equivalents of azide.
Mentions: The X-band EPR spectra of Cu-Ao(AA11) at 150 K in the absence and presence of azide are shown in Fig. 5. The azide-free structure exhibits a near-axial spectral envelope revealing a singly-occupied molecular orbital at copper with significant d(x2-y2) character. Accurate simulation of the Cu-Ao(AA11) spectrum could be achieved in the parallel region with gz = 2.27 and ∣Az∣ = 157 G (0.0166 cm−1, Supplementary Table 2), placing Cu-Ao(AA11) squarely within the Peisach-Blumberg classification of a type 2 copper centre.25 Given the lack of resolution in the perpendicular region, accurate simulation of the Ax,y and of gx,y values was not possible, however an overall fit to the spectral envelope could only be achieved by introducing a degree of rhombicity, with gx = 2.03 and gy = 2.10. It is notable that there is some resolution of superhyperfine coupling to ligating nitrogen atoms observable in the perpendicular region (~15 G, 0.0014 cm−1, 43 MHz) with coupling constants typical for coordination to copper(II) by sp2 hybridised nitrogen atoms.26 Addition of excess sodium azide causes a shift in the EPR spectrum of Cu-Ao(AA11) to gx = gy = 2.06, gz = 2.24 and ∣Az∣ = 175 G (0.018 cm−1, Fig. 5, Supplementary Table 2) demonstrating that azide coordinates directly to the copper ion, accompanied by a shift in coordination geometry symmetry towards axial. A comparison of the known EPR parameters for the different LPMO classes and substrates, using the standard axial type 2 copper parameters in AA9 as a reference (Supplementary Table 3) shows that AA10 enzymes which are active on chitin demonstrate a reduced ∣Az∣ value and some rhombicity in perpendicular g values. AA11 enzymes have type 2 copper ∣Az∣ values and rhombicity in g values, thereby lying somewhat in-between AA9 and AA10 in terms of their EPR spectroscopic features, commensurate with the structural variations in the active sites of the different classes.

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