Ole Golten

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are exceptionally powerful small redox enzymes that master the controlled generation and productive use of potentially damaging hydroxyl radicals in what is essentially a H ₂ O ₂ -driven peroxygenase reaction. We have used ancestral sequence reconstruction and enzyme resurrection to unravel evolutionary steps leading to this exceptional catalytic ability. Real-time monitoring of copper reoxidation and amino acid radical formation showed evolutionary improvement of both the capacity to avoid futile turnover of H ₂ O ₂ and the ability to scavenge damaging radicals resulting from such turnover through a hole hopping pathway. Through mutational studies of ancestral LPMOs, we show that adoption of an extant-like conformation of residues in the hole hopping pathway yields improvements in redox robustness to near-extant levels. These results show how selective pressure imposed by the need for generating a highly oxidizing intermediate is a key driver of metalloenzyme evolution, involving large parts of the enzyme, well beyond the catalytic center.

Lytic polysaccharide monooxygenases (LPMOs) play a critical role in the depolymerization of recalcitrant polysaccharides, such as chitin, making them of interest in biotechnological applications. These interfacial enzymes are also of great chemical interest because of their unique monocopper catalytic center and their ability to activate high energy C–H bonds. This report investigates the structural and electronic changes at the copper (Cu) site of an LPMO, SmAA10A, upon binding of its chitin substrate, utilizing a suite of spectroscopic and computational methods. Herein, we present the first reported X-ray Absorption (XAS) and Emission (XES) spectroscopic data on substrate-bound LPMO. By comparing the Cu(II) and Cu(I) states of SmAA10A in both the chitin-bound and unbound states, we provide insights into the structural adjustments facilitating substrate specificity and productive catalytic turnover. Our results indicate a substrate binding-induced conformational change in Cu(I) site geometry and concurrent modulations to the electronic structure, which prime the enzyme for targeted C–H activation with an H₂O₂ co-substrate. This work offers an atomistic understanding of interaction dynamics between the LPMO Cu site and the chitin substrate, advancing our knowledge of LPMO functionality and substrate specificity.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that hydroxylate recalcitrant polysaccharides such as cellulose. Like other redox enzymes, LPMOs face challenges in handling the reactive oxygen species generated at their active site, which must be controlled to prevent off-pathway reactions that lead to enzyme inactivation. In the case of LPMOs, oxidative damage may be self-reinforcing because free copper released from damaged catalytic centers will promote abiotic redox reactions that generate reactive oxygen species. Here we show that some members of a widely spread family of carbohydrate-binding modules (CBM2s) have evolved the ability to bind copper and that this ability is exclusively found in CBM2s that are appended to LPMOs. We show that the copper site in these CBM2s protects the LPMO from inactivation both by scavenging free copper, preferably Cu(I), and by interacting directly with the reduced catalytic copper site of the LPMO, thus preventing the enzyme from engaging in off-pathway reactions. These effects are demonstrated by studies on the redox stability of a series of engineered LPMO variants as well as AlphaFold3 models of the CBM2-containing enzymes. Interestingly, the copper site and the cellulose-binding surface are located on different sides of these CBM2s, enabling a mode of action in which the CBM inhibits potentially damaging LPMO activity in the absence of substrate, while such inhibition would be relieved upon binding of the CBM2 to cellulose. These findings show that CBMs have biologically relevant functions beyond carbohydrate-binding and reveal a mechanism for substrate-dependent regulation of LPMO reactivity.

Enzymes known as lytic polysaccharide monooxygenases (LPMOs) are mono‐copper polysaccharide‐degrading peroxygenases that engage in several on‐ and off‐pathway redox reactions involving O₂ and H₂O₂. Herein, we show that the known metalloenzyme inhibitor cyanide inhibits reductive activation of LPMOs by binding to the LPMO‐Cu(II) state and that the degree of inhibition depends on the concentrations of the polysaccharide substrate, the reductant and H₂O₂. Importantly, this analysis revealed differences between fungal NcAA9C and bacterial SmAA10A, which have different secondary copper coordination spheres. These differences were also highlighted by the observation that phosphate, a commonly used buffer ion, strongly inhibits NcAA9C while not affecting reactions with SmAA10A. The results provide insight into LPMO inhibition and catalysis and highlight pitfalls in the analysis thereof.

Lytic Polysaccharide Monooxygenases (LPMOs) catalyze the oxidative depolymerization of polysaccharides at a monocopper active site, that is coordinated by the so-called histidine brace. In the past, this motif has sparked considerable interest, mostly due to its ability to generate and stabilize highly oxidizing intermediates during catalysis. We used a variety of advanced EPR techniques, including Electron Nuclear Double Resonance (ENDOR), Electron Spin Echo Envelope Modulation (ESEEM) and Hyperfine Sublevel Correlation (HYSCORE) spectroscopy in combination with isotopic labelling (¹⁵N, ²H) to characterize the active site of the bacterial LPMO SmAA10A over a wide pH range (pH 4.0–pH 12.5). At elevated pH values, several ligand modifications are observed, including changes in the H_xO ligand coordination, but also regarding the protonation state of the histidine brace. At pH > 11.5, the deprotonation of the two remote nitrogen nuclei of the imidazole moieties and of the terminal amine is observed. These deprotonations are associated with major electronic changes, including increased σ-donor capabilities of the imidazolates and an overall reduced interaction of the deprotonated amine function. This observation highlights a potentially more significant role of the imidazole ligands, particularly for the stabilization of potent oxidants during turnover. The presented study demonstrates the application of advanced EPR techniques for a thorough characterization of the active site in LPMOs, which ultimately sets a foundation for and affords an outlook on future applications characterizing reaction intermediates.

Lytic polysaccharide monooxygenases (LPMOs) are enzymes that catalyze the oxidative depolymerization of polysaccharides through activation of strong C-H bonds. Their active site is composed of a copper ion that is coordinated by the so-called histidine brace, consisting of two histidine residues that coordinate the metal ion in a T-shaped arrangement. The unique structure of this motif and its suggested ability to generate and stabilize highly oxidizing intermediates has sparked considerable interest, leading to various hypotheses on its role for LPMOs and LPMO-inspired biomimetic complexes. To gain detailed insights into the electronic and geometric structure of the histidine brace and test its chemical variability, we have used advanced electron paramagnetic resonance (EPR) techniques, in combination with isotopic labelling (15N, 2H) of the AA10 LPMO SmAA10A over a wide pH range (pH 4.0 - pH 12.5), in which the monocopper site remains intact and the protein does not exhibit permanent damage. Between pH 4 and 11.5, Electron Nuclear Double Resonance (ENDOR) spectroscopy identifies and discriminates between coordinating waters and hydroxo ions (pKa1 = 9.65) at an open-site of the histidine brace. Above pH 11.5, deprotonation of the two remote nitrogen nuclei of the coordinating imidazole moieties and of the coordinating N-terminal amine function was observed via ENDOR, Electron Spin Echo Envelope Modulation (ESEEM) and Hyperfine Sublevel Correlation (HYSCORE) spectroscopies. This is associated with major electronic changes in the histidine brace, including increased σ-donor capabilities of the imidazolates and an overall reduced interaction of the deprotonated amine function with the copper center. This observation highlights the possible larger role of the imidazole moieties, potentially stabilizing potent oxidants during turnover. The associated spectroscopic and electronic changes of the LPMO are discussed in reference with functional biomimetic complexes. The presented study demonstrates the application of advanced EPR techniques for a thorough characterization of the active site in LPMOs, which ultimately sets a foundation for and affords an outlook on future applications characterizing reaction intermediates.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, using a single copper cofactor bound in a conserved histidine brace with a more variable second coordination sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif, whereas other cellulose-active AA10s have an Arg-Glu-Phe motif. To shine a light on the impact of this variation, we generated single, double, and triple mutations changing the His²¹⁶-Gln²¹⁹-Tyr²²¹ motif in cellulose- and chitin-oxidizing MaAA10B toward Arg-Glu-Phe. These mutations generally reduced enzyme performance due to rapid inactivation under turnover conditions, showing that catalytic fine-tuning of the histidine brace is complex and that the roles of these second sphere residues are strongly interconnected. Studies of copper reactivity showed remarkable effects, such as an increase in oxidase activity following the Q219E mutation and a strong dependence of this effect on the presence of Tyr at position 221. In reductant-driven reactions, differences in oxidase activity, which lead to different levels of in situ generated H₂O₂, correlated with differences in polysaccharide-degrading ability. The single Q219E mutant displayed a marked increase in activity on chitin in both reductant-driven reactions and reactions fueled by exogenously added H₂O₂. Thus, it seems that the evolution of substrate specificity in LPMOs involves both the extended substrate-binding surface and the second coordination sphere.

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein’s surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Biological conversion of plant biomass depends on peroxygenases and peroxidases acting on insoluble polysaccharides and lignin. Among these are cellulose- and hemicellulose-degrading lytic polysaccharide monooxygenases (LPMOs), which have revolutionized our concept of biomass degradation. Major obstacles limiting mechanistic and functional understanding of these unique peroxygenases are their complex and insoluble substrates and the hard-to-measure H₂O₂ consumption, resulting in the lack of suitable kinetic assays. We report a versatile and robust electrochemical method for real-time monitoring and kinetic characterization of LPMOs and other H₂O₂-dependent interfacial enzymes based on a rotating disc electrode for the sensitive and selective quantitation of H₂O₂ at biologically relevant concentrations. The H₂O₂ sensor works in suspensions of insoluble substrates as well as in homogeneous solutions. Our characterization of multiple LPMOs provides unprecedented insights into the substrate specificity, kinetics, and stability of these enzymes. High turnover and total turnover numbers demonstrate that LPMOs are fast and durable biocatalysts.

Lytic polysaccharide monooxygenases (LPMOs) are powerful monocopper enzymes that can activate strong C-H bonds through a mechanism that remains largely unknown. Herein, we investigated the role of a conserved glutamine/glutamate in the second coordination sphere. Mutation of the Gln in NcAA9C to Glu, Asp, or Asn showed that the nature and distance of the headgroup to the copper fine-tune LPMO functionality and copper reactivity. The presence of Glu or Asp close to the copper lowered the reduction potential and decreased the ratio between the reduction and reoxidation rates by up to 500-fold. All mutants showed increased enzyme inactivation, likely due to changes in the confinement of radical intermediates, and displayed changes in a protective hole-hopping pathway. Electron paramagnetic resonance (EPR) and X-ray absorption spectroscopic (XAS) studies gave virtually identical results for all NcAA9C variants, showing that the mutations do not directly perturb the Cu(II) ligand field. DFT calculations indicated that the higher experimental reoxidation rate observed for the Glu mutant could be reconciled if this residue is protonated. Further, for the glutamic acid form, we identified a Cu(III)-hydroxide species formed in a single step on the H₂O₂ splitting path. This is in contrast to the Cu(II)-hydroxide and hydroxyl intermediates, which are predicted for the WT and the unprotonated glutamate variant. These results show that this second sphere residue is a crucial determinant of the catalytic functioning of the copper-binding histidine brace and provide insights that may help in understanding LPMOs and LPMO-inspired synthetic catalysts.