LPMO activity monitored by a turbidimetric assay
Turbidimetry has been recently employed to screen the cellulolytic activity of a fungal LPMO towards phosphoric acid-swollen cellulose (PASC), which represents a disordered, amorphous form of cellulose. This screening assay measured the decrease in the optical density of the substrate after a defined incubation time of 360 min at 50 °C in microwell plates [38]. Here, we adapt this procedure into a continuous, turbidimetric assay to measure the time-dependent conversion of PASC by a cellulose-active LPMO.
Initially, we established the relation between PASC concentration and the loss of transmitted light intensity. The optical attenuation was linear up to a concentration of 1.4 mg mL−1 PASC (Fig. 1a). These measurements were performed under constant stirring to prevent the settling of particles in the suspension. In the standard assay, we employed a concentration of PASC (0.8 mg mL−1) that provided a stable baseline and a low background signal from the light scattering of larger substrate particles in the suspension. The molar concentration of PASC was 24.7 µM assuming an average chain length of 200 glucose units [39]. However, the particle distribution of PASC is not homogenous, which affects the depolymerization kinetics as discussed later. The reaction was started by injecting an LPMO-containing stock solution, which also contained the reducing agent. In experiments using H2O2 as the cosubstrate, the stock solution was added before addition of the H2O2. The optical density of the PASC suspension was continuously monitored at a wavelength of 620 nm, which was previously used for the turbidimetric measurement of cellulase activity [40].
Binding of LPMO to PASC
In the following experiments, we employed LPMO9C from Neurospora crassa (NcLPMO9C; UniProt accession number Q7SHI8), which is active on cellulose, hemicelluloses and soluble oligosaccharides [9, 11, 41]. This LPMO contains a family 1 carbohydrate-binding module (CBM1) which is fused to the catalytic domain via a lengthy linker peptide of 82 amino acids. In the first set of experiments, we employed 1 mM ascorbate, which is a commonly used concentration in LPMO conversion assays. The assay was started after 240 s by the addition of a relatively high concentration of LPMO (3 µM) to achieve a fast assay. Unexpectedly, this led to an instant increase in optical density within the mixing time (Fig. 1b). For both the reduced and the oxidized NcLPMO9C, the optical density increased linearly with the enzyme concentration, but the observed increase was approximately three times higher for the reduced LPMO (Fig. 1c). The same increase in optical density was also observed when mixing ascorbate and LPMO under anaerobic conditions, demonstrating that this phase represents a non-catalytic reaction (Fig. 1d). Control experiments in the absence of PASC did not show detectable absorbance changes for all employed LPMO concentrations.
The fact that the reduced LPMO showed a higher increase in optical density than its oxidized form under both aerobic and anaerobic conditions suggests that the rapid initial increase in optical density is due to improved substrate binding. Previous binding experiments demonstrated a higher substrate affinity of NcLPMO9C to PASC when the active site was in the reduced state [42]. In this study, the presence of ascorbate increased both the binding affinity and the binding capacity to PASC approximately twice [42]. A similar observation was made for the binding of LPMO9E from Myceliophthora thermophila to soluble oligosaccharides [43]. The binding of different substrate chains by the catalytic domain and the CBM1 under reducing conditions may lead to a “cross-linking” of PASC fibres and may thereby increase the optical density.
Ascorbate-driven LPMO activity
Following the initial, very rapid increase in optical density, a second phase showing an attenuation of the signal was observed in assays containing LPMO and ascorbate (Fig. 1b). The decrease in optical density indicates the degradation of the PASC by the LPMO. To confirm catalysis, we mixed NcLPMO9C with ascorbate in an anaerobic glove box (Fig. 1d) in the absence of any oxygen species. We observed the first phase of the reaction (binding of the LPMO to PASC), but found that the second, catalytic phase was completely suppressed. In the following, LPMO activity is expressed as the relative change in optical density per min. The rates were calculated from the linear slopes of the catalytic phase to avoid substrate depletion at the end of the experiment. An important and unexpected observation from these experiments is that almost similar reaction rates were obtained for different LPMO concentrations (Fig. 1e, blue triangles). The observed uncoupling of catalyst concentration and reaction rate—a fourfold increase of enzyme concentration correlated to a 25% increase of the activity—points towards a rate-limiting factor in the overall reaction. One reason could be the concentration of the reductant ascorbate, which was applied in a 1 mM concentration. We, therefore, varied the ascorbate concentration for 3 µM NcLPMO9C (Fig. 1f). Initial rates calculated from these batch conversions demonstrated a strong correlation between activity and ascorbate concentration (Fig. 1e, black circles). A previous study that employed the bacterial SmLPMO10A and chitin as the substrate showed a clear dependency of the LPMO reaction rate on the reductant concentration, with an apparent KM for ascorbate of 2 µM [44]. However, it is also well documented that ascorbate can reduce O2 to H2O2 under commonly employed reaction conditions [24, 28]. Thus, providing a higher ascorbate concentration in the assays is likely to release higher amounts of H2O2, which can act as a cosubstrate for LPMO. To test whether the availability of H2O2 was the rate-limiting factor in the measurements, we replicated the activity assays in the presence of catalase (final concentration: 2000 U mL−1 at pH 6) to scavenge most of the formed H2O2. Under these conditions, we still observed the initial increase in optical density upon addition of LPMO, indicating that substrate binding of the LPMO was not compromised by the catalase. However, the subsequent catalytic reaction was clearly, but not fully suppressed in the presence of catalase (Fig. 1f, dashed lines).
Interaction of NcLPMO9C with NcCDHIIA
We also initiated LPMO activity with cellobiose dehydrogenase (CDH), which is a proposed native interaction partner of LPMOs in wood-decaying fungi [20, 24]. CDHs oxidize cellobiose or soluble cello-oligosaccharides in an FAD-dependent reaction and reduce the LPMO active site via a dedicated, flexible cytochrome domain [21]. Reduced CDHs also have a low, FAD-dependent oxidase activity [45, 46], which can support LPMO activity through the slow release of H2O2 [33]. We used NcCDHIIA (UniProt accession number Q7RXM0), the main secreted CDH in N. crassa [47], to activate NcLPMO9C in the PASC turbidity assays (Fig. 2a). The activity of LPMO in this reaction setup was strictly dependent on the presence of cellobiose as CDH substrate (Additional file 1: Figure S1). NcCDHIIA in combination with cellobiose induced moderate LPMO activity, which was dependent on the applied NcCDHIIA concentration. The observed rates were approximately one order of magnitude lower than those obtained with ascorbate as LPMO-reductant (Figs. 2b vs 1e). Catalase (2000 U mL−1) completely inhibited the reaction at a low, 0.5 µM concentration of NcCDHIIA, while at higher concentrations a weak LPMO activity was observed, possibly reflecting the incomplete H2O2 removal by the catalase. The obvious inhibition of the turbidimetric PASC assay by catalase at low CDH concentrations indicates that H2O2 was predominantly used as cosubstrate by NcLPMO9C. Since both NcLPMO9C and NcCDHIIA feature a CBM1 that binds to cellulose, the spatial proximity of the two enzymes during catalysis, which is required for the electron transfer between both enzymes, may also provide a locally increased H2O2 concentration in the vicinity of the heterogeneous substrate.
To further probe the effect of H2O2 on CDH-driven LPMO activity, we used commercial glucose oxidase (GOX) from Aspergillus niger for the in situ generation of H2O2. GOX in combination with glucose and LPMO did not lead to changes in the optical density (Fig. 2c), demonstrating that an LPMO-specific reductant is required to induce activity. For LPMO reduction a low, 0.5 µM concentration of NcCDHIIA in combination with 10 mM cellobiose was added. Under these conditions, the addition of GOX led to a rate enhancement that depended on the applied GOX activity and, therefore, also on the amount of produced H2O2. At high GOX activities, a fast, initial attenuation of the optical density was followed by a slower phase of signal decay. This indicates a rapid deactivation of NcLPMO9C at high GOX concentrations, possibly due to H2O2-induced oxidation of the copper-coordinating amino acids [26]. Such deactivation effects were recently observed for a bacterial LPMO, which was rapidly deactivated when the H2O2 supply exceeded the enzyme’s capability to convert the cosubstrate [33]. The pronounced rate acceleration upon addition of GOX in the presence of a low, 0.5 µM concentration of NcCDHIIA indicated that not the availability of reducing equivalents, but the H2O2 concentration was the rate-limiting factor in these reactions.
To verify that the observed increase in activity upon H2O2 addition detected by turbidimetry corresponds to the formation of oxidized oligosaccharide products, MALDI-MS measurements were performed on the soluble fraction of the reaction mixtures. The formation of products was followed in reactions containing PASC, LPMO, CDH and lactose and in related reactions spiked several times with H2O2 during the course of the incubation (Fig. 3). C4 oxidized products, which are typical products of the NcLPMO9C reaction [9], were detected in the form of sodium adducts of C4 ketones and geminal diols. Small amounts of native (unoxidized) oligosaccharides, e.g. Glc3, Glc4 and Glc5, were also present in control samples containing only PASC, CDH and lactose. Such products may also occur during the LPMO action due to a weak hydrolytic background [48]. While absolute quantitation cannot be achieved by MALDI-MS, the changes in the ratio of unoxidized and oxidized oligosaccharides between the individual conditions clearly indicated the boosting effect of H2O2 on the action of NcLPMO9C (Fig. 3b–d).
The high resolving power and high mass accuracy of the FT-ICR MS allowed us to unambiguously assign different carbohydrate molecules and their adduct state. For example, we were able to clearly distinguish between Glc(n)(K+) and Glc(n)Gemdiol(Na+) adducts, which differ only by 0.02 Da. The mass measurements can also provide indirect proof whether the LPMO generates C1 or C4 oxidized products. C1 oxidation leads to the formation of sugar lactones, which undergo conversion into aldonic acids. The acidic products are then preferentially detected in the form of salt (sodium or potassium), charged by an additional alkali metal cation (Na+ or K+) [27, 34]. On the other hand, C4 oxidizing LPMOs create keto/gemdiol forms, which are not forming salts and are present only as single alkali metal cation charged masses. Since we have not detected aldonic acids in any of the reaction mixtures and only detected gemdiols, we can conclude that the NcLPMO9C indeed generated C4 oxidation products.
The peroxygenase reactivity of LPMO
To determine the H2O2 consumption rate by LPMO, we tested the reactivity of NcLPMO9C with H2O2 by titrating aliquots of H2O2 to reactions containing 0.8 mg mL−1 PASC, 3 µM LPMO and 2 mM ascorbate. In these experiments, H2O2 was added to the reaction every 90 s using three different concentrations (20, 40 or 80 µM per addition). The total change in the reaction volume due to the addition of H2O2 was less than 3% in all assays. The addition of H2O2 to reduced LPMO caused an immediate decrease in optical density, which points towards a fast consumption of H2O2. This reaction was much faster than the reference reaction without H2O2 (Fig. 4a). The substrate conversion rate could not be determined because it was as fast or faster than the mixing time of the cuvette (ca. 10 s). However, doubling the amount of added H2O2 also doubled the observed change in optical density. For all titrations, approximately 350–400 µM of H2O2 was required to reach maximal observable changes, corresponding to approximately 0.2 units of optical density. Addition of H2O2 or LPMO beyond this lower limit did not induce further changes in the optical density of the PASC suspension. Control experiments in which either LPMO or reductant were omitted did not show any changes in the optical density of the PASC suspension (Additional file 1: Figure S2). Likewise, the titration of H2O2 to oxidized NcLPMO9C had no observable effect on the optical density of the PASC (Additional file 1: Figure S2).
To correlate the observed substrate degradation with the cosubstrate consumption, we followed the depletion of H2O2 using electrochemical detection of H2O2 (Fig. 4b). These assays were carried out at a larger volume of 12 mL in a stirred electrochemical cell to avoid exceeding consumption of H2O2 by the electrode. Titration of 40 µM H2O2 to reactions containing only PASC or only LPMO showed a stable, H2O2 concentration-dependent decrease of the measured current. The addition of H2O2 to oxidized LPMO resulted in slightly lower currents, indicating H2O2 depletion through a background reaction. Under these conditions, no turbidimetric changes of PASC were observed (Additional file 1: Figure S2) showing that this futile reaction did not induce observable catalytic events. The addition of H2O2 to reactions containing 2 mM ascorbate (Fig. 4b, magenta line) led to a slow depletion of H2O2, possibly via reduction of the H2O2 [49, 50]. Upon titration of H2O2 to a reaction containing ascorbate, LPMO and PASC, no detectable increase in the H2O2 concentration was observed, showing that H2O2 was rapidly consumed in this experiment (Fig. 4b, red line). This is a clear indication that the consumption of the cosubstrate by the system occurred within the response time of the electrochemical sensor, which was approximately 3 s. After 9 H2O2 additions, corresponding to a total added H2O2 concentration of 360 µM, a built-up of H2O2 was observed. This concentration coincides with the required H2O2 concentration that induced maximal changes in optical density of PASC in degradation assays carried out under comparable conditions (Fig. 4a, red line). Doubling the concentration of added H2O2 to 80 µM per addition (Fig. 4b, blue line) resulted in notable signal spikes after 4–5 additions (320–400 µM), which compares well to the experiments shown in Fig. 4a which employed the same H2O2 addition rate. Taken together, these experiments demonstrate fast consumption of H2O2 by an LPMO-dependent reaction and connect the observed absorbance changes to the consumption of H2O2. The visual change that accompanied the degradation of PASC by LPMO upon titration with 40 µM H2O2 is shown in Fig. 4c. The images suggest that, to a large extent, NcLPMO9C preferentially targeted finely dispersed, amorphous PASC while bigger particles remained largely intact at the end of the reaction. The heterogeneity of the substrate may also explain why the reaction levelled off at a certain optical density.
Substrate binding of LPMO during H2O2-mediated PASC degradation
To gain further insight into the binding of LPMO to PASC, we monitored the fraction of free NcLPMO9C during the titration of reduced LPMO with 10 aliquots of 40 µM H2O2. Samples of 50 µL were regularly withdrawn from this reaction and the supernatants analysed by SDS-PAGE after centrifugation (Fig. 5a). Incubation of LPMO with PASC in absence of reductant reduced the concentration of soluble LPMO by 50%, indicating binding of the other 50% of LPMO to the substrate. Addition of ascorbate to this reaction instantly increased the fraction of bound enzyme to 71%. This compares well to the observed changes in optical density in Fig. 1b, which showed a higher signal change for the reduced LPMO when compared to the oxidized enzyme. The fraction of free enzyme gradually increased upon titration with H2O2 (Fig. 5a). Quantitative assessment of PASC by weight determination (Fig. 5b) showed that notable substrate degradation occurred only in samples containing ascorbate together with LPMO. Addition of H2O2 to this mixture led to a notably higher PASC degradation than observed in the presence of ascorbate alone. In this reaction, approximately 20% of the PASC initially present in the assay was solubilized by the LPMO. In the same reaction, the optical density of PASC decreased by ca. 45% (from 0.47 to 0.21 optical density at 620 nm). Thus, part of the observed absorbance changes may be a result of PASC modification rather than solubilization, e.g. via the introduction of oxidized ends, or the release of insoluble oligomers. Results obtained from bacterial or fungal LPMOs previously showed that only approximately 50% of the total introduced oxidized ends were found on soluble oligomers, while the remaining modifications occurred on the insoluble fraction [33].