Discovery and characterization of a new family of lytic polysaccharide monooxygenases

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.

Figure 1

Structure of typical AA9 and active sites of AA9 and AA10. (a) Overall structure of AA9 from Thermoascus aurantiacus with the active site copper shown as a sphere and active site residues shown as sticks, (b) schematic representations of the Cu active sites observed in AA9 and AA10 structures.

Figure 2

“Module walking” to discover new LPMOs.

Figure 3

Copper binding affinity and oxidative activity of Ao(AA11). (a) ITC thermogram of Zn²⁺ displacement by Cu²⁺ from the active site of Ao(AA11). (b) MALDI-TOF analysis of Ao(AA11) action on squid-pen chitin. DPn_al = aldonic acid, DPn₋₂ = oxidation from R-OH to R=O, (measured MW). DP5/DP5₋₂ +Na⁺ (1056.4, 1054.4), DP5_al +Na⁺ (1072.4), DP5_al⁻ +2Na⁺ (1094.4), DP6/DP6₋₂ +Na⁺ (1259.5, 1257.5), DP6_al +Na⁺ (1275.5), DP6_al⁻ +2Na⁺ (1297.5), DP7/DP7₋₂ +Na⁺ (1462.6, 1460.6), DP7_al +Na⁺ (1478.6), DP7_al⁻ +2Na⁺ (1500.6), DP8/DP8₋₂ +Na⁺ (1665.6, 1663.6), DP8_al +Na⁺ (1681.6), DP8_al⁻ +2Na⁺ (1703.6), DP9/DP9₋₂ +Na⁺ (1868.7, 1866.7), DP9_al +Na⁺ (1884.7), DP10/DP10₋₂ +Na⁺ (2071.8, 2069.8), DP10_al +Na⁺ (2087.8), DP10_al⁻ +2Na⁺ (2109.8). Magnified region of DP6 is shown in Supplementary Fig. 3.

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.

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.