C L E A N E N E R G Y T R A N S I T I O N A N D S U S T A I N A B L E T E C H N O L O G I E S

S C I E N T I F I C H I G H L I G H T S

1 1 0 H I G H L I G H T S 2 0 2 3 I

Crystallography reveals the complex role of histidine brace copper enzymes in biomass utilisation

Lytic polysaccharide monooxygenases (LPMOs) were discovered as enzymatic tools to boost the bioconversion of recalcitrant biomass. Since then, diverse LPMO-like proteins similar in structure, but with diverse functions, have emerged. X-ray crystallography underpins that the AA16 enzyme boosts activity of other LPMOs, while apparently not being an LPMO itself.

Transition from a fossil-based society to a more sustainable one drives the full valorisation of lignocellulose-rich agricultural and forestry side-streams for the production of biofuels, biomaterials and biochemicals. The enzyme- driven degradation of cellulose and hemicellulose to fermentable monosaccharides is an essential step, in which lytic polysaccharide monooxygenases (LPMOs) are key. LPMOs belonging to the auxiliary activities (AA) class of the carbohydrate-active enzymes (CAZymes [1]) are copper-dependent enzymes that degrade polysaccharides oxidatively. To explore the AA diversity in nature and improve enzyme formulations, new AA families with enigmatic functions need further investigation.

The AA class contains redox enzymes assisting other enzymes in complex carbohydrate degradation and is divided in sequence-based families, of which AA9- AA11 and AA13-AA17 contain LPMOs. The structural elucidation of numerous LPMOs has unveiled a common, immunoglobulin-like fold and a recurring active site motif, the histidine brace, formed by the N-terminus and two histidine side chains binding the active site copper [2]. A flat surface, often containing an aromatic

residue running parallel to it, allows cellulose-active LPMOs to interact with the surface of cellulose fibres. Initially, LPMOs were thought to be monooxygenases (using O2 as co-substrate), but now it is increasingly accepted that H2O2 is a preferred co-substrate, in most cases in a peroxygenase reaction (Figure 88) [3]. It is widely acknowledged that the priming reaction of Cu(II) reduction to Cu(I) by an external electron source, which can be a small molecule like ascorbic acid (Asc) or a protein, initiates the catalytic cycle. In the absence of polysaccharide substrates, many LPMOs have oxidase activity, producing H2O2 from O2.

MtAA16A is a member of the AA16 LPMO family, one of the more recently discovered cellulolytic LPMO families [4], and was isolated from the fungus Myceliophthora thermophila, a source of many robust enzymes used for lignocellulose degradation in the biorefinery. Against expectations, MtAA16A didn t cleave common plant cell wall polysaccharides like cellulose, hemicellulose, chitin, pectin, oligosaccharides or even their mixtures, though redox activity could be shown against small substrates which can be used for indirect colorimetric assays.

This puzzling finding prompted structure determination by X-ray crystallography using beamline ID30A-3. In this work, the MtAA16A structure, the first experimental structure for an AA16 enzyme, could be determined to a maximum resolution of 2.65 Å (PDB accession: 7ZE9). The structure shows an ordered and copper-bound histidine brace active site with very similar geometry to known active LPMOs (Figure 89). In contrast to the flat surface usually observed in cellulose-active LPMOs, a small pocket is found next to the copper active-site formed by Hic1, Tyr27, and other residues. This pocket can help rationalise that MtAA16A shows activity on small

Fig. 88: Schematic view of an LPMO peroxygenase reaction with cellulose in the presence of reductant (e ) and H2O2.The star indicates an oxidised product in cellulose.

The structure shown as an example is of LsAA9A (PDB: 7PQR) and illustrates the immunoglobulin-like fold and the histidine brace copper binding site, which also includes

a Tyr. In the initial priming reaction, Cu(II) (orange) is transformed to Cu(I) (blue).