The copper active site of CBM33 polysaccharide oxygenases.

Glyn R Hemsworth,Paul H Walton,Robbert Q Kim,Edward J Taylor,Sally J Lewis,Gideon J Davies,Johan P Turkenburg,Rebecca C Gregory,Alison Parkin

doi:10.1021/ja402106e

Abstract

The capacity of metal-dependent fungal and bacterial polysaccharide oxygenases, termed GH61 and CBM33, respectively, to potentiate the enzymatic degradation of cellulose opens new possibilities for the conversion of recalcitrant biomass to biofuels. GH61s have already been shown to be unique metalloenzymes containing an active site with a mononuclear copper ion coordinated by two histidines, one of which is an unusual τ-N-methylated N-terminal histidine. We now report the structural and spectroscopic characterization of the corresponding copper CBM33 enzymes. CBM33 binds copper with high affinity at a mononuclear site, significantly stabilizing the enzyme. X-band EPR spectroscopy of Cu(II)-CBM33 shows a mononuclear type 2 copper site with the copper ion in a distorted axial coordination sphere, into which azide will coordinate as evidenced by the concomitant formation of a new absorption band in the UV/vis spectrum at 390 nm. The enzyme’s three-dimensional structure contains copper, which has been photoreduced to Cu(I) by the incident X-rays, confirmed by X-ray absorption/fluorescence studies of both aqueous solution and intact crystals of Cu-CBM33. The single copper(I) ion is ligated in a T-shaped configuration by three nitrogen atoms from two histidine side chains and the amino terminus, similar to the endogenous copper coordination geometry found in fungal GH61.

Highlights

Controlled degradation of abundant biomass is a sine qua non for the future success of bioethanol production.[1−5] In this regard, finding a means of overcoming the recalcitrance of cellulosic, lignocellulosic, or chitinotic materials either by chemical or by enzymatic methods is a major objective
We have demonstrated that a CBM33 from Bacillus amyloliquefaciens binds copper(II) with a Kd ≈ 6 nM
From X-band EPR spectroscopy and azide binding studies, we can infer that Cu-BaCBM33 is a mononuclear type 2 copper enzyme, but where the copper(II) ion has a distorted axial geometry, possibly a distorted square planar configuration akin to that seen in structure of Cu−Zn superoxide dismutase

Summary

■ INTRODUCTION

Controlled degradation of abundant biomass is a sine qua non for the future success of bioethanol production.[1−5] In this regard, finding a means of overcoming the recalcitrance of cellulosic, lignocellulosic, or chitinotic materials either by chemical or by enzymatic methods is a major objective. This was shown by Li et al, who identified conserved tyrosine residues on the α-helical L2 loop in polysaccharide oxygenases and on residues near the copper active site (Figure 8).[21] The residues are in different positions on the binding face when compared to CBM33s, and probably interact with the cellulose substrate in a somewhat less directional way than the potential hydrogenbonding interactions seen in CBM33 This is commensurate with a wider range of oxidation sites on cellulose that is observed with GH61 oxidative action, especially type 3 PMOs,[21] and that oxidation does occur at the exposed C1−H bonds on cellulose, as appears to be the case in CBM33. Notwithstanding these caveats, a structural basis for the known differences in mechanisms of action of CBM33s and GH61s does emerge without the need to introduce significant post-translational modifications in CBM33s, and may well be a reliable basis for understanding differences in reactivity

■ CONCLUSIONS

■ EXPERIMENTAL PROCEDURES

■ ACKNOWLEDGMENTS

■ REFERENCES