Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components

Abstract

The enzymatic degradation of recalcitrant plant biomass is one of the key industrial challenges of the 21st century. Accordingly, there is a continuing drive to discover new routes to promote polysaccharide degradation. Perhaps the most promising approach involves the application of "cellulase-enhancing factors," such as those from the glycoside hydrolase (CAZy) GH61 family. Here we show that GH61 enzymes are a unique family of copper-dependent oxidases. We demonstrate that copper is needed for GH61 maximal activity and that the formation of cellodextrin and oxidized cellodextrin products by GH61 is enhanced in the presence of small molecule redox-active cofactors such as ascorbate and gallate. By using electron paramagnetic resonance spectroscopy and single-crystal X-ray diffraction, the active site of GH61 is revealed to contain a type II copper and, uniquely, a methylated histidine in the copper's coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

Fig. 1.

PASC deconstruction by TaGH61A, AoBG, and various concentrations of gallate. 5 g/L PASC was incubated with the indicated gallate and TaGH61A concentrations at an AoBG concentration of 20 mg/g cellulose, 3-d reaction at 50 °C in 50 mM sodium acetate, pH 5, and 1 mM MnSO₄. GH61 cleaved cellulose into cellodextrin, which was converted to glucose by AoBG and quantified by refractive index detection HPLC to calculate PASC conversion.

Fig. 2.

MALDI-TOF-MS analysis of permethylated products from 5 g/L PASC, 13 mg/g cellulose TaGH61A, and 3 mM gallate in 25 mM triethylammonium acetate and 2 mM CaCl₂ incubated at pH 5.4 for 22 h. (Inset) Expanded view of spectra in the DP5 range.

Fig. 3.

The structure of the active site and the EPR spectrum of Cu-TaGH61. (A) 3D structure of the primary metal site as observed in a T. aurantiacus GH61A crystal soaked in 10 mM CuNO₃ for 30 min. Maps shown are the maximum likelihood/σ_A weighted 2F_obs − F_calc density (contoured at 0.4 electrons per ų) in blue, and the ACORN unbiased direct methods Emap contoured at 1.1 electrons per ų (3.3σ) in red. Also shown is a small molecule modeled as a PEG fragment that bonds in the equatorial position. Unmodeled density (at ∼1/5 the Cu site) lies ∼1.6 Å from the Cu; it is unclear whether this residual electron density is a counterion or a low occupancy of a copper ion in a different position due to oxidation state or protonation state differences. Images were drawn with CCP4Mg (34). (B) X-band EPR spectrum (140 K) of copper-loaded TaGH61A in 10 mM acetate buffer and ∼15% (vol/vol) glycerol. (C) Schematic diagram of the copper site in GH61, depicting histidine brace.

Fig. 4.

Copper-loaded TaGH61A is catalytically active as seen by PACE (A) and MALDI-TOF (B) analyses of saccharide products formed during the reaction of 4 mg/g cellulose of TaGH61A with 5 g/L PASC in the presence of 10 mM ascorbate for 1 h at 50 °C at pH 5, with 25 mM triethylammonium acetate. TaGH61A had previously been demetallated and, where indicated, loaded with Cu(II). A shows the spectrum of saccharide products with an unmodified reducing end, and B shows the full spectrum of DP6 products. The product profile is similar to that from the reaction of TaGH61 with undefined metal load with PASC using gallate as the reducing agent (as seen in Fig. 2).