Molecular mechanism of the chitinolytic peroxygenase reaction

Abstract

This PDF file includes:1. Complete experimental and computational details 2. Supplementary results 3. Supplementary discussion 4. List of supplementary figures 5. Supplementary Figure 1 to 26 6. Abbreviations list 7. Supplementary references 8. QM/MM optimized xyz coordinates of states 1-9 chromatography (HPAEC) coupled to pulsed amperometric detection (PAD) using a Dionex Bio-LC equipped with a CarboPac PA1 column as previously described. 3 To quantify A2 ox , a standard was produced in-house by treating chitobiose (Megazymes) with a chitooligosaccharide oxidase (ChitO) from Fusarium graminearum, which yields 100% conversion of chitobiose to chitobionic acid. 2,4 All chromatograms were recorded using Chromeleon 7.0 software.Chitin binding assay. The capacity of SmAA10A-WT and mutants thereof to bind β-chitin was tested by suspending 10 mg/mL of substrate in sodium phosphate buffer (50 mM, pH 7.0) in a total volume of 600 µL in 2 mL Eppendorf tubes. Reactions were started by the addition of SmAA10A (1 µM final concentration) and were incubated and stirred in an Eppendorf Comfort Thermomixer (at 40 °C, 1000 rpm). Samples were taken (100 µL) after 15, 30, 60, 120 and 240 min and immediately filtrated using a 96-well filter plate (Millipore) operated with a vacuum manifold to obtain the unbound protein fraction.In order to assess the percentage of bound proteins to the substrate, control samples with only enzyme and buffer were included, representing the maximum quantity of protein present in the samples (i.e. 100% unbound). The protein concentration in each sample was determined using the Bradford assay (Bio-Rad, Munich, Germany).H2O2 consumption experiments. H2O2 consumption by SmAA10A-WT and mutants thereof was measured according to a previously described protocol 5 using conditions that were slightly different from the standard reaction conditions described above: in order to be able to monitor the H2O2 consumption within a reasonable timescale the enzyme concentration had to be reduced and EDTA was added to reduce the background reaction of free metals-catalyzed H2O2 reduction (see Figure S6). After optimization, a standard reaction mixture contained the LPMO (50 nM) and H2O2 (100 µM) and EDTA (50 µM), without or with b-chitin (10 g.L -1 ), in sodium phosphate buffer (50 mM, pH 7.0), and the mixtures were incubated at 40 °C in a thermomixer (1000 rpm). The reactions were initiated by addition of AscA (20 µM final concentration). At regular intervals (t = 3, 6, 9, 12, 30 and 60 min), 70 µL of the reaction mixture was sampled, filtered as described above and 25 µL of the filtrate was mixed with 75 µL of a pre-mix of HRP (5 U.mL -1 final concentration) and Amplex® Red (ThermoFisher) (100 µM final concentration) in sodium phosphate buffer (50 mM pH 7.0). H2O2 concentrations waere then determined spectrophotometrically by measuring the absorbance at 540 nm in a microtiter plate reader.An H2O2 standard curve was prepared in the same conditions. Bioinformatics analysis. The sequence of the chitin-binding protein f...

Controlled depolymerization of cellulose by light-driven lytic polysaccharide oxygenases

“…The correlations between H 2 O 2 availability and LPMO activity described above suggest that this priming reduction is not rate-limiting. This is supported by stopped-flow kinetics showing fast (4.2 × 10 5 M −1 s −1 ) and full reduction of an AA10 LPMO when using as little as 5 µM AscA 23 . The situation may be different when using the Chl/light system, without added AscA.…”

“…The distribution between these different pathways will depend on kinetics of the different reactions. Available kinetic studies predict that, in the presence of a suitable substrate, the productive substrate hydroxylation pathway will be favored 23,25 .…”

“…The reaction with H 2 O 2 is best described as that of a peroxygenase (R-H + H 2 O 2 → R-OH + H 2 O), where the oxygen atom, the two protons and the reducing equivalents are supplied simultaneously in the form of H 2 O 2 to an activated LPMO (i.e., the reduced LPMO-Cu(I) state). Subsequently, several experimental studies have confirmed the efficiency of H 2 O 2 -driven LPMO reactions for different LPMO/substrate systems [23][24][25][26][27][28][29] . Furthermore, modeling studies have demonstrated the feasibility of the peroxygenase reaction 23,[30][31][32][33] .…”

“…Of note, however, the rate-limiting step in this photobiocatalytic system likely resides in the thermodynamically challenging oxidation of water (E 0 ox = −1. 23 (Supplementary Fig. 6).…”

The H2O2-dependent activity of a fungal lytic polysaccharide monooxygenase investigated with a turbidimetric assay

“…Even if the K M -value of NcLPMO9C for ascorbate would be 50-times higher, the high 0.5-6 mM ascorbate concentration present in our assays should still provide sufficiently saturating conditions to achieve maximal turnover. The reduction of the active site by ascorbate is not the rate-limiting step in the overall LPMO reaction at high ascorbate concentrations [53] and providing more reducing equivalents should not exert a boosting effect on the LPMO catalysis. From experiments with the H 2 O 2 scavenger catalase, we conclude that the H 2 O 2 generated from the oxidation reaction of O 2 by ascorbate is preferentially used as cosubstrate by the NcLPMO9C for the degradation of the cellulose substrate.…”