Paul H. Walton

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

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 coppers coordination sphere, thus providing an innovative paradigm in bioinorganic enzymatic catalysis.

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

Lytic polysaccharide mono-oxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They currently attract much attention due to their potential use in biomass conversion, notably in the production of biofuels. Past work has 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 3-D structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active centre featuring the histidine brace active site, but is distinct in terms of its active site details and its EPR spectroscopy. The new AA11 family expands the LPMO clan with the potential to broaden both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.

Recent insights into copper-containing lytic polysaccharide mono-oxygenases.

Recently the role of oxidative enzymes in the degradation of polysaccharides by saprophytic bacteria and fungi was uncovered, challenging the classical model of polysaccharide degradation of being solely via a hydrolytic pathway. 3D structural analyses of lytic polysaccharide mono-oxygenases of both bacterial AA10 (formerly CBM33) and fungal AA9 (formerly GH61) enzymes revealed structures with β-sandwich folds containing an active site with a metal coordinated by an N-terminal histidine. Following some initial confusion about the identity of the metal ion it has now been shown that these enzymes are copper-dependent oxygenases. Here we assess recent developments in the academic literature, focussing on the structures of the copper active sites. We provide critical comparisons with known small-molecules studies of copper-oxygen complexes and with copper methane monoxygenase, another of natures powerful copper oxygenases.

Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes.

Lignocellulose degradation mechanisms across the Tree of Life

Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.

The copper active site of CBM33 polysaccharide oxygenases.

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.

Lytic Polysaccharide Monooxygenases in Biomass Conversion

The derivation of second-generation biofuels from non-edible biomass is viewed as crucial for establishing a sustainable bio-based economy for the future. The inertness of lignocellulosic biomass makes its breakdown for conversion into fuels and other compounds a challenge. Enzyme cocktails can be utilized in the bio-refinery for lignocellulose deconstruction but until recently their costs were regarded as high. Lytic polysaccharide monooxygenases (LPMOs) offer tremendous promise for further process improvements owing to their ability to boost the activity of biomass-degrading enzyme consortia. Combining data from multiple disciplines, progress has been made in understanding the biochemistry of LPMOs. We review the academic literature in this area and highlight some of the key questions that remain.

Nickel‐Assisted Carbon–Fluorine Bond Activation of 2,4,6‐Trifluoropyrimidine: Synthesis of New Pyrimidine and Pyrimidinone Derivatives

Rapid and regioselective activation of the C-F bond of 2,4,6-trifluoropyrimidine occurs on reaction with [Ni(cod)(2)] (cod=1,5-cyclooctadiene) in the presence of PEt(3) to give 1, which can be converted into complex 2, containing a further N(3)-metalated pyrimidin-4-one unit. The novel pyrimidin-4-one 3 is released on protonation of 2.

The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains.

Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases

Significance Activation of the O-O bond in dioxygen is difficult but fundamental in biology. Nature has evolved several strategies to achieve this, often including copper as an enzyme cofactor. Copper-dependent enzymes usually use more than one metal to activate O2 by multielectron reduction, but recently it was discovered that cellulose and chitin degrading polysaccharide monooxygenase enzymes use only a single Cu center for catalysis, in a reaction that is of great interest to the biofuel industries. To understand this reactivity, we have determined the solution structures of both the reduced and oxidized Cu site, and determined experimentally and computationally how this site is capable of facile O2 activation by a thermodynamically difficult one-electron reduction, via an inner-sphere Cu-superoxide intermediate. Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9–11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.