Molecular modelling and site-directed mutagenesis provide insight into saccharide pyruvylation by the Paenibacillus alvei CsaB enzyme.

Pyruvylation is a biologically versatile but mechanistically unexplored saccharide modification. 4,6-Ketal pyruvylated N-acetylmannosamine within bacterial secondary cell wall polymers serves as a cell wall anchoring epitope for proteins possessing a terminal S-layer homology domain trimer. The pyruvyltransferase CsaB from Paenibacillus alvei served as a model to investigate the structural basis of the pyruvyltransfer reaction by a combination of molecular modelling and site-directed mutagenesis together with an enzyme assay using phosphoenolpyruvate (PEP; donor) and synthetic ß-D-ManNAc-(1 → 4)-α-D-GlcNAc-diphosphoryl-11-phenoxyundecyl (acceptor). CsaB protein structure modelling was done using Phyre2 and I-Tasser based on the partial crystal structure of the Schizosaccharomyces pombe pyruvyltransferase Pvg1p and by AlphaFold. The models informed the construction of twelve CsaB mutants targeted at plausible PEP and acceptor binding sites and KM and kcat values were determined to evaluate the mutants, indicating the importance of a loop region for catalysis. R148, H308 and K328 were found to be critical to PEP binding and insight into acceptor binding was obtained from an analysis of Y14 and F16 mutants, confirming the modelled binding sites and interactions predicted using Molecular Operating Environment. These data lay the basis for future mechanistic studies of saccharide pyruvylation as a novel target for interference with bacterial cell wall assembly.

Mutations Increasing Cofactor Affinity, Improve Stability and Activity of a Baeyer-Villiger Monooxygenase.

The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer-Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C T m) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.

Inhibition of the Peroxygenase Lytic Polysaccharide Monooxygenase by Carboxylic Acids and Amino Acids.

Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in fungi, and catalyze the oxidative degradation of polysaccharides such as cellulose. Despite their name, LPMOs possess a dominant peroxygenase activity that is reflected in high turnover numbers but also causes deactivation. We report on the influence of small molecules and ions on the activity and stability of LPMO during catalysis. Turbidimetric and photometric assays were used to identify LPMO inhibitors and measure their inhibitory effect. Selected inhibitors were employed to study LPMO activity and stability during cellulose depolymerization by HPLC and turbidimetry. It was found that the fungal metabolic products oxalic acid and citric acid strongly reduce LPMO activity, but also protect the enzyme from deactivation. QM calculations showed that the copper atom in the catalytic site could be ligated by bi- or tridentate chelating compounds, which replace two water molecules. MD simulations and QM calculations show that the most likely inhibition pattern is the competition between the inhibitor and reducing agent in the oxidized Cu(II) state. A correlation between the complexation energy and the IC50 values demonstrates that small, bidentate molecules interact strongest with the catalytic site copper and could be used by the fungus as physiological effectors to regulate LPMO activity.

A cytochrome b-glucose dehydrogenase chimeric enzyme capable of direct electron transfer.

The development of third generation biosensors depends on the availability of direct electron transfer (DET) capable enzymes. A successful strategy is to fuse a cytochrome domain to an enzyme to fulfil the function of a built-in redox mediator between the catalytic center and the electrode. In this study, we fused the cytochrome domain of Neurospora crassa CDH IIA (NcCYT) N-terminally to glucose dehydrogenase from Glomerella cingulata (GcGDH) to generate the chimeric enzyme NcCYT-GcGDH in a large amount for further studies. Heterologous expression in P. pastoris and chromatographic purification resulted in 1.8 g of homogeneous chimeric enzyme. Biochemical and electrochemical characterization confirmed that the chimeric enzyme is catalytically active, able to perform interdomain electron transfer (IET) and direct electron transfer (DET) via the fused cytochrome domain. The midpoint redox potential of the fused b-type cytochrome is 91 mV vs. SHE at pH 6.5 and the specific current obtained on a porous graphite electrode is 2.3 µA cm-2. The high current obtained on this simple, unmodified electrode at a rather low redox potential is a promising starting point for further optimization. The high yield of NcCYT-GcGDH and its high specific activity supports the application of the chimeric enzyme in bioelectrocatalytic applications.

Insights into the H2 O2 -driven catalytic mechanism of fungal lytic polysaccharide monooxygenases.

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H2 O2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H2 O2 is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H2 O2 as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single 'priming' electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H2 O2 -driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H2 O2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H2 O2 cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu+ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H2 O2 for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu+ state of LPMOs, which may prevent the formation of uncoupled side reactions.

Protein Conformational Change Is Essential for Reductive Activation of Lytic Polysaccharide Monooxygenase by Cellobiose Dehydrogenase.

Large-scale protein domain dynamics and electron transfer are often associated. However, as protein motions span a broad range of time and length scales, it is often challenging to identify and thus link functionally relevant dynamic changes to electron transfer in proteins. It is hypothesized that large-scale domain motions direct electrons through a FAD and a heme b cofactor of the fungal cellobiose dehydrogenase (CDH) enzymes to the type-II copper center (T2Cu) of the polysaccharide-degrading lytic polysaccharide monooxygenases (LPMOs). However, as of yet, domain motions in CDH have not been linked formally to enzyme-catalyzed electron transfer reactions. The detailed structural features of CDH, which govern the functional conformational landscapes of the enzyme, have only been partially resolved. Here, we use a combination of pressure, viscosity, ionic strength, and temperature perturbation stopped-flow studies to probe the conformational landscape associated with the electron transfer reactions of CDH. Through the use of molecular dynamics simulations, potentiometry, and stopped-flow spectroscopy, we investigated how a conserved Tyr99 residue plays a key role in shaping the conformational landscapes for both the interdomain electron transfer reactions of CDH (from FAD to heme) and the delivery of electrons from the reduced heme cofactor to the LPMO T2Cu. Our studies show how motions gate the electron transfer within CDH and from CDH to LPMO and illustrate the conformational landscape for interdomain and interprotein electron transfer in this extracellular fungal electron transfer chain.

Improved spectrophotometric assay for lytic polysaccharide monooxygenase.

BACKGROUND: The availability of a sensitive and robust activity assay is a prerequisite for efficient enzyme production, purification, and characterization. Here we report on a spectrophotometric assay for lytic polysaccharide monooxygenase (LPMO), which is an advancement of the previously published 2,6-dimethoxyphenol (2,6-DMP)-based LPMO assay. The new assay is based on hydrocoerulignone as substrate and hydrogen peroxide as cosubstrate and aims toward a higher sensitivity at acidic pH and a more reliable detection of LPMO in complex matrices like culture media. RESULTS: An LPMO activity assay following the colorimetric oxidation of hydrocoerulignone to coerulignone was developed. This peroxidase activity of LPMO in the presence of hydrogen peroxide can be detected in various buffers between pH 4-8. By reducing the substrate and cosubstrate concentration, the assay has been optimized for minimal autoxidation and enzyme deactivation while maintaining sensitivity. Finally, the optimized and validated LPMO assay was used to follow the recombinant expression of an LPMO in Pichia pastoris and to screen for interfering substances in fermentation media suppressing the assayed reaction. CONCLUSIONS: The biphenol hydrocoerulignone is a better substrate for LPMO than the monophenol 2,6-DMP, because of a ~ 30 times lower apparent K M value and a 160 mV lower oxidation potential. This greatly increases the measured LPMO activity when using hydrocoerulignone instead of 2,6-DMP under otherwise similar assay conditions. The improved activity allows the adaptation of the LPMO assay toward a higher sensitivity, different buffers and pH values, more stable assay conditions or to overcome low concentrations of inhibiting substances. The developed assay protocol and optimization guidelines increase the adaptability and applicability of the hydrocoerulignone assay for the production, purification, and characterization of LPMOs.

Interaction between Cellobiose Dehydrogenase and Lytic Polysaccharide Monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are ubiquitous oxidoreductases, facilitating the degradation of polymeric carbohydrates in biomass. Cellobiose dehydrogenase (CDH) is a biologically relevant electron donor in this process, with the electrons resulting from cellobiose oxidation being shuttled from the CDH dehydrogenase domain to its cytochrome domain and then to the LPMO catalytic site. In this work, we investigate the interaction of four Neurospora crassa LPMOs and five CDH cytochrome domains from different species using computational methods. We used HADDOCK to perform protein-protein docking experiments on all 20 combinations and subsequently to select four complexes for extensive molecular dynamics simulations. The potential of mean force is computed for a rotation of the cytochrome domain relative to LPMO. We find that the LPMO loops are largely responsible for the preferred orientations of the cytochrome domains. This leads us to postulate a hybrid version of NcLPMO9F, with exchanged loops and predicted altered cytochrome binding preferences for this variant. Our work provides insight into the possible mechanisms of electron transfer between the two protein systems, in agreement with and complementary to previously published experimental data.

A fast and sensitive activity assay for lytic polysaccharide monooxygenase.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMO) release a spectrum of cleavage products from their polymeric substrates cellulose, hemicellulose, or chitin. The correct identification and quantitation of these released products is the basis of MS/HPLC-based detection methods for LPMO activity. The duration, effort, and intricate analysis allow only specialized laboratories to measure LPMO activity in day-to-day work. A spectrophotometric assay will simplify the screening for LPMO in culture supernatants, help monitor recombinant LPMO expression and purification, and support enzyme characterization. RESULTS: Based on a newly discovered peroxidase activity of LPMO, we propose a fast, robust, and sensitive spectrophotometric activity assay using 2,6-dimethoxyphenol (2,6-DMP) and H2O2. The fast enzymatic assay (300 s) consists of 1 mM 2,6-DMP as chromogenic substrate, 100 µM H2O2 as cosubstrate, and an adequate activity of LPMO in a suitable buffer. The high molar absorption coefficient of the formed product coerulignone (ε469 = 53,200 M-1 cm-1) makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5-50 mg L-1. CONCLUSIONS: The activity assay based on 2,6-DMP detects a novel peroxidase activity of LPMO. This activity can be accurately measured and used for enzyme screening, production, and purification, and can also be applied to study binding constants or thermal stability. However, the assay has to be used with care in crude extracts, because other enzymes such as laccase or peroxidase will interfere with the assay. We also want to stress that the peroxidase activity is a homogeneous reaction with soluble substrates and should not be correlated to heterogeneous LPMO activity on polymeric substrates.

Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells.

Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6.5. Beneficial mutation sites were investigated by semi-rational and combinatorial mutagenesis. Fourteen variants were characterised in detail to evaluate changes of the kinetic constants. Mutations increasing thermostability were distributed over the entire structure. Among them, T383I showed a 2.6-fold increased half-life by preventing the loss of the T2 copper through unfolding of a loop. Mutations affecting the pH-dependence cluster around the T1 copper and categorise in three types of altered pH profiles: pH-type I changes the monotonic decreasing pH profile into a bell-shaped profile, pH-type II describes increased specific activity below pH 6.5, and pH-type III increased specific activity above pH 6.5. Specific activities of the best variants were up to 5-fold higher (13 U mg-1) than BaL WT at pH 7.5.

Extracellular electron transfer systems fuel cellulose oxidative degradation.

Ninety percent of lignocellulose-degrading fungi contain genes encoding lytic polysaccharide monooxygenases (LPMOs). These enzymes catalyze the initial oxidative cleavage of recalcitrant polysaccharides after activation by an electron donor. Understanding the source of electrons is fundamental to fungal physiology and will also help with the exploitation of LPMOs for biomass processing. Using genome data and biochemical methods, we characterized and compared different extracellular electron sources for LPMOs: cellobiose dehydrogenase, phenols procured from plant biomass or produced by fungi, and glucose-methanol-choline oxidoreductases that regenerate LPMO-reducing diphenols. Our data demonstrate that all three of these electron transfer systems are functional and that their relative importance during cellulose degradation depends on fungal lifestyle. The availability of extracellular electron donors is required to activate fungal oxidative attack on polysaccharides.