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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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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. DPnal = aldonic acid, DPn−2 = oxidation from R-OH to R=O, (measured MW). DP5/DP5−2 +Na+ (1056.4, 1054.4), DP5al +Na+ (1072.4), DP5al− +2Na+ (1094.4), DP6/DP6−2 +Na+ (1259.5, 1257.5), DP6al +Na+ (1275.5), DP6al− +2Na+ (1297.5), DP7/DP7−2 +Na+ (1462.6, 1460.6), DP7al +Na+ (1478.6), DP7al− +2Na+ (1500.6), DP8/DP8−2 +Na+ (1665.6, 1663.6), DP8al +Na+ (1681.6), DP8al− +2Na+ (1703.6), DP9/DP9−2 +Na+ (1868.7, 1866.7), DP9al +Na+ (1884.7), DP10/DP10−2 +Na+ (2071.8, 2069.8), DP10al +Na+ (2087.8), DP10al− +2Na+ (2109.8). Magnified region of DP6 is shown in Supplementary Fig. 3.
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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. DPnal = aldonic acid, DPn−2 = oxidation from R-OH to R=O, (measured MW). DP5/DP5−2 +Na+ (1056.4, 1054.4), DP5al +Na+ (1072.4), DP5al− +2Na+ (1094.4), DP6/DP6−2 +Na+ (1259.5, 1257.5), DP6al +Na+ (1275.5), DP6al− +2Na+ (1297.5), DP7/DP7−2 +Na+ (1462.6, 1460.6), DP7al +Na+ (1478.6), DP7al− +2Na+ (1500.6), DP8/DP8−2 +Na+ (1665.6, 1663.6), DP8al +Na+ (1681.6), DP8al− +2Na+ (1703.6), DP9/DP9−2 +Na+ (1868.7, 1866.7), DP9al +Na+ (1884.7), DP10/DP10−2 +Na+ (2071.8, 2069.8), DP10al +Na+ (2087.8), DP10al− +2Na+ (2109.8). Magnified region of DP6 is shown in Supplementary Fig. 3.

Mentions: Previous studies on AA94,5 and AA1019,20 have shown that these LPMOs are copper-dependent enzymes which bind copper with tight binding constants. Accordingly, isothermal titration calorimetry on apo-Ao(AA11) exhibited the same tight binding of copper with binding too tight to be determined accurately and thus estimated to have a KD < 1 nM (data not shown). Accurate determination of the copper binding constant was achieved by displacement calorimetry21 in which a weaker binding and kinetically labile metal, Zn2+ (KD 8 μM at pH 5 with Ao(AA11), data not shown), was first bound to apo-Ao(AA11) and then the Cu2+ binding titrated competitively, allowing accurate determination of a dissociation constant, KD, for Cu-Ao(AA11) of 790 ±150 pM at pH 5, Fig. 3a.


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

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

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. DPnal = aldonic acid, DPn−2 = oxidation from R-OH to R=O, (measured MW). DP5/DP5−2 +Na+ (1056.4, 1054.4), DP5al +Na+ (1072.4), DP5al− +2Na+ (1094.4), DP6/DP6−2 +Na+ (1259.5, 1257.5), DP6al +Na+ (1275.5), DP6al− +2Na+ (1297.5), DP7/DP7−2 +Na+ (1462.6, 1460.6), DP7al +Na+ (1478.6), DP7al− +2Na+ (1500.6), DP8/DP8−2 +Na+ (1665.6, 1663.6), DP8al +Na+ (1681.6), DP8al− +2Na+ (1703.6), DP9/DP9−2 +Na+ (1868.7, 1866.7), DP9al +Na+ (1884.7), DP10/DP10−2 +Na+ (2071.8, 2069.8), DP10al +Na+ (2087.8), DP10al− +2Na+ (2109.8). Magnified region of DP6 is shown in Supplementary Fig. 3.
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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. DPnal = aldonic acid, DPn−2 = oxidation from R-OH to R=O, (measured MW). DP5/DP5−2 +Na+ (1056.4, 1054.4), DP5al +Na+ (1072.4), DP5al− +2Na+ (1094.4), DP6/DP6−2 +Na+ (1259.5, 1257.5), DP6al +Na+ (1275.5), DP6al− +2Na+ (1297.5), DP7/DP7−2 +Na+ (1462.6, 1460.6), DP7al +Na+ (1478.6), DP7al− +2Na+ (1500.6), DP8/DP8−2 +Na+ (1665.6, 1663.6), DP8al +Na+ (1681.6), DP8al− +2Na+ (1703.6), DP9/DP9−2 +Na+ (1868.7, 1866.7), DP9al +Na+ (1884.7), DP10/DP10−2 +Na+ (2071.8, 2069.8), DP10al +Na+ (2087.8), DP10al− +2Na+ (2109.8). Magnified region of DP6 is shown in Supplementary Fig. 3.
Mentions: Previous studies on AA94,5 and AA1019,20 have shown that these LPMOs are copper-dependent enzymes which bind copper with tight binding constants. Accordingly, isothermal titration calorimetry on apo-Ao(AA11) exhibited the same tight binding of copper with binding too tight to be determined accurately and thus estimated to have a KD < 1 nM (data not shown). Accurate determination of the copper binding constant was achieved by displacement calorimetry21 in which a weaker binding and kinetically labile metal, Zn2+ (KD 8 μM at pH 5 with Ao(AA11), data not shown), was first bound to apo-Ao(AA11) and then the Cu2+ binding titrated competitively, allowing accurate determination of a dissociation constant, KD, for Cu-Ao(AA11) of 790 ±150 pM at pH 5, Fig. 3a.

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