Oxidative cleavage of polysaccharides by mono-copper ... · 1 1 2 3 Supplementary Information for 4...

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Supplementary Information for 3

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Oxidative cleavage of polysaccharides by mono-copper 5

enzymes depends on H2O2 6

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Bastien Bissaro1,2*

, Åsmund K. Røhr2, Gerdt Müller

2, Piotr Chylenski

2, Morten Skaugen

2, 9

Zarah Forsberg2, Svein J. Horn

2, Gustav Vaaje-Kolstad

2, and Vincent G.H. Eijsink

2* 10

11 1. INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France 12 2. Faculty of Chemistry, Biotechnology and Food Science, Norwegian University of 13 Life Sciences (NMBU), P.O. Box 5003, N-1432 Aas, Norway 14 15

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*Correspondence to: [email protected], [email protected] 17

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This PDF file includes: 21

22

1. List of supplementary figures 23

2. Supplementary Figure 1 to 19 24

3. Full abbreviations list 25

4. Supplementary references 26

27

28

Nature Chemical Biology: doi:10.1038/nchembio.2470

mailto:[email protected]

mailto:[email protected]

2

SUPPLEMENTARY RESULTS 29

1. List of supplementary figures 30

31

Supplementary Fig. 1 Reductive activation of ScLPMO10C by superoxide. 32

Supplementary Fig. 2 Impact of initial exogenous H2O2 on cellulose oxidation 33

efficiency under various conditions. 34

Supplementary Fig. 3 The effect of H2O2 on LPMO activity. 35

Supplementary Fig. 4 Control reactions to check for occurrence of copper-catalyzed 36

Fenton-type chemistry. 37

Supplementary Fig. 5 Screening conditions to probe the “priming reduction” hypothesis. 38

Supplementary Fig. 6 H2O2 consumption at high AscA concentration. 39

Supplementary Fig. 7 Activation of ScLPMO10C-catalyzed degradation of Avicel by 40

AA3-mediated in situ generation of H2O2. 41

Supplementary Fig. 8 Product profile (HPAEC-PAD) obtained after degradation of 42

cellulose by ScLPMO10C in reactions carried out in aerobic or O2-free conditions, in the 43

presence or absence of initial exogenous H2O2 (100 µM). 44

Supplementary Fig. 9 Probing the origin of oxygen introduced into the polysaccharide 45

chain by ScLPMO10C using H218

O2. 46

Supplementary Fig. 10 Assessing the competition between H2O2 and O2 by using low 47

concentrations of H218

O2. 48

Supplementary Fig. 11 Probing the origin of oxygen introduced into the polysaccharide 49

chain by the chitin-active AA10 CBP21 using H218

O2. 50

Supplementary Fig. 12 Probing the origin of the oxygen introduced into the 51

polysaccharide chain by the fungal cellulose-active PcLPMO9D using H218

O2. 52

Supplementary Fig. 13 Inhibition of ScLPMO10C-catalyzed degradation of Avicel by 53

by the H2O2–consuming HRP/Amplex Red system. 54

Supplementary Fig. 14 Putative reaction mechanisms for polysaccharide oxidation by 55

LPMOs using H2O2 or O2 as co-substrate. 56

Supplementary Fig. 15 Study of ScLPMO10C inactivation by the Chl/AscA system in 57

the dark or in the light. 58

Supplementary Fig. 16 Semi-quantitative distribution of selected modifications 59

observed in samples of pretreated LPMO. 60

Supplementary Fig. 17 Identification of residues modified during LPMO inactivation. 61

Supplementary Fig. 18 Location of oxidative modifications in ScLPMO10C. 62

Supplementary Fig. 19 Inactivation of ScLPMO10C by AscA/H2O2 and the protective 63

role of the substrate. 64

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2. Supplementary figures 66

67

68 69

Supplementary Figure 1 Reductive activation of ScLPMO10C by superoxide. (a) 70

Time-course for the release of aldonic acid products from Avicel (10 g.L-1

) by 71

ScLPMO10C (1 µM) in Tris-HCl (pH 8.0, 50 mM). Reactions were initiated by the 72

addition of KO2 (280 µM final concentration, approximately) or AscA (1 mM final 73

concentration). The KO2 stock solution was preprared in dried and N2-flushed DMSO. 74

Due to the instability of KO2 in protic solvents its actual concentration once added in the 75

reaction mixture cannot be controlled. For multiple additions, KO2 (280 µM, final added 76

concentration), alone or with ScLPMO10C (1 µM final added concentration) were added 77

right after each sampling . The oxidation rate measured for KO2-driven reactions was on 78

average 70-fold lower than for reactions driven by AscA (note that the scales of the two 79

y-axes differ by one order of magnitude). Note that superoxide will spontaneously 80

disproportionate to H2O2, which can have a negative effect on LPMO activity if the H2O2 81

concentrations become too high (as demonstrated further below); the occurrence of 82

enzyme inactivation is illustrated by the beneficial effect of repetitively adding 83

ScLPMO10C on product yield. (b) Quantity of soluble aldonic acids released from 84

Avicel (10 g.L-1

) by ScLPMO10C (0.5 µM) after 18 h incubation of reactions prepared in 85

sodium phosphate (50 mM, pH 7.0) and fueled by the system xanthine (XTH, 500 86

µM)/xanthine oxidase (XOD, 0.1 to 100 to mU.mL-1

). A control reaction using AscA 87

(500 µM) instead of XTH/XOD was included. All reactions were carried out at 40 °C, 88

under magnetic stirring. Before product quantification, cello-oligosaccharides were 89

hydrolyzed by TfCel5A, yielding oxidized products with a degree of polymerization of 2 90

and 3 [GlcGlc1A, (Glc)2Glc1A], the quantities of which were summed up to yield the 91

concentration of oxidized sites. The data shown in this Figure are from a single 92

experiment. 93

94


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95 96

Supplementary Figure 2 Impact of initial exogenous H2O2 on cellulose oxidation 97

efficiency under various conditions. The three panels show time courses for the release 98

of aldonic acid products from Avicel (10 g.L-1

) by ScLPMO10C (0.5 µM) fueled by the 99

system Chlorophyllin (Chl, 500 µM)/light + AscA (1 mM) (left), only water (middle) or 100

only AscA (1 mM) (right), as indicated in the Figure. Reactions were carried out in 101

sodium phosphate buffer (50 mM, pH 7.0) at 40 °C, under magnetic stirring and exposed 102

to visible light (I =25% Imax, approx. 42 W.cm-2

) for Chl/light+AscA reactions or 103

incubated in the dark for the other reactions. Before product quantification, cello-104

oligosaccharides were hydrolyzed by TfCel5A, yielding oxidized products with a degree 105

of polymerization of 2 and 3 [GlcGlc1A, (Glc)2Glc1A], summed up to yield the 106

concentration of oxidized sites. The data shown in this Figure are from a single 107

experiment. 108

The study with light was based on work by Cannella et al.1. Compared to this 109

previous work, we used higher light dosages which led to high initial rates and full 110

enzyme inactivation prior to the first sampling point. In this case, addition of exogeneous 111

H2O2 had a negative effect. Notably, these conditions were used to study the LPMO 112

inactivation phenomenon and its underlying mechanism, as described below (See 113

Supplementary Figs. 15-18). 114

115


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116 117

Supplementary Figure 3 The effect of H2O2 on LPMO activity. The panels show 118

time-courses for the release of aldonic acid products from Avicel (10 g.L-1

) by 0.5 M 119

ScLPMO10C (a; zoom-in view in b), 0.5 µM PcLPMO9D (d, e),0.5 µM ScLPMO10B 120

(g, h) or from β-chitin (10 g.L-1

) by 0.5 µM CBP21 (j, k) in the presence of different 121


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initial concentrations of exogenous H2O2 (0-1000 µM) and AscA (1 mM). Panels c, f, i 122

and l show how the apparent initial LPMO rate depends on the H2O2 concentration for 123

ScLPMO10C, PcLPMO9D, ScLPMO10B and CBP21, respectively. The relative increase 124

in apparent initial rate (calculated on the basis of product formation after 2, 3, 3 and 10 125

min for ScLPMO10C, PcLPMO9D, ScLPMO10B and CBP21, respectively) compared to 126

the reference reaction (without H2O2) is provided on the secondary axis. All reactions 127

were carried out in sodium phosphate buffer (50 mM, pH 7.0) at 40 °C, under magnetic 128

stirring, in the dark. Before product quantification, cello-oligosaccharides were 129

hydrolyzed by TfCel5A, yielding oxidized products with a degree of polymerization of 2 130

and 3 [GlcGlc1A, (Glc)2Glc1A], summed up to yield the concentration of oxidized sites. 131

Solubilized chito-oligosaccharides were hydrolyzed with a chitobiase, yielding 132

chitobionic acid as the only oxidized product. Error bars show ± s.d. (n = 3). 133

It is worth mentioning that full length enzymes have been used in this experiment 134

(and throughout the entire study). ScLPMO10C has a carbohydrate-binding module 135

(CBM) belonging to family 2 (CBM2), whereas PcLPMO9D and ScLPMO10B do not 136

have any CBM. The chitin-active CBP21 does not have a CBM but binds strongly to 137

chitin. 138

It is important to note that the concentrations of H2O2 used here are high relative 139

to what the LPMOs need and can handle. Thus, in most cases, already at the first 140

measuring point, product levels reflect a trade-off between enzyme activity and enzyme 141

inactivation, meaning that the apparent initial rates depicted in panels c, f, i and l are no 142

true initial rates. The enzymes show large differences in this respect. Such differences are 143

well illustrated by the differences between the progress curves for the reactions with 144

ascorbic acid only, i.e. reactions where H2O2 is produced continuoulsy and at the same 145

rate. Experimentally, it is very difficult to control and assess the fraction of activated 146

LPMO and the actual concentration of H2O2. Notably, when using the present conditions, 147

H2O2 can be generated from O2 by the LPMO or by AscA and may also be further 148

reduced to water by AscA. 149 150


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151 152

Supplementary Figure 4 Control reactions to check for occurrence of copper-153

catalyzed Fenton-type chemistry. The enzyme, ScLPMO10C (0.5 µM) (a) or CBP21 154

(0.5 µM) (b), was replaced by Cu(II)SO4 (0.5 µM) in the presence of different initial 155

concentrations of exogenous H2O2 (0-1000 µM). All reactions were carried out in sodium 156

phosphate buffer (50 mM, pH 7.0) with Avicel (10 g.L-1

) (A) or β-chitin (10 g.L-1

) (b), 157

incubated for 60 min at 40 °C in glass vials under magnetic stirring, and initiated by 158

addition of AscA (1 mM). The figure shows the product profile of (oxidized) cello-159

oligosaccharides analyzed by HPAEC-PAD (a) and of oxidized chito-oligosaccharides 160

analyzed by HILIC-UV (b). Glc1A, aldonic acid of glucose (Glc); GlcNAc1A, aldonic 161

acid of N-acetyl-glucosamine (GlcNAc). The data shown in this Figure are from a single 162

experiment. 163


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164 165

Supplementary Figure 5 Screening conditions to probe the “priming reduction” 166

hypothesis. The graph shows time-courses for the release of aldonic acid products from 167

Avicel (10 g.L-1

) by ScLPMO10C (0.5 µM) in the presence (100 µM, left side) or 168

absence (right side) of initial exogenous H2O2 and at different concentrations of AscA 169

(0.5-100 µM). All reactions were carried out in sodium phosphate buffer (50 mM, pH 170

7.0) at 40 °C, under magnetic stirring, in the dark. Before product quantification, 171

celloligosaccharides were hydrolyzed by TfCel5A, yielding oxidized products with a 172

degree of polymerization of 2 and 3 [GlcGlc1A, (Glc)2Glc1A], summed up to yield the 173

concentration of oxidized sites. In the absence of H2O2, one can barely observe oxidized 174

products during the first 30 min for AscA concentrations between 0.5 and 10 µM. In the 175

presence of H2O2, 10 µM of AscA seems to be the lowest concentration allowing 176

detection of soluble oxidized products. Note that the amount of oxidized products 177

detected in the presence of H2O2 (left side) is higher than the amount of added AscA. The 178

data shown in this Figure are from a single experiment. 179


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180 181

Supplementary Figure 6 H2O2 consumption at high AscA concentration. The 182

reaction mix contained ScLPMO10C (0.5 µM), H2O2 (100 µM) and Avicel (10 g.L-1

). 183

Reactions were carried out in sodium phosphate buffer (50 mM, pH 7.0) at 40 °C, under 184

magnetic stirring. In the control reaction (blue line) ScLPMO10C-Cu(II) was replaced by 185

Cu(II)SO4 (0.5 µM). The reactions were initiated by addition of ascorbic acid (1 mM). 186

An initial concentration of 100 µM of exogenous H2O2 was chosen since at this 187

concentration ScLPMO10C maintains activity during at least the first hour and is not 188

quickly inactivated (see Supplementary Fig. 3). Error bars show ± s.d. (n = 3). 189

190


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191 192

Supplementary Figure 7 Activation of ScLPMO10C-catalyzed degradation of 193

Avicel by AA3-mediated in situ generation of H2O2. Panel (a) is identical to Fig. 1c 194

from the main article and shows time-courses for the release of aldonic acids from Avicel 195

(10 g.L-1

) by ScLPMO10C (0.5 µM) in the presence of different concentrations of 196

glucose oxidase from Aspergillus niger (AnGOX, 0-236 ng.mL-1

) and glucose (15 mM). 197

Reactions (200 µL total volume) were carried out in sodium phosphate buffer (50 mM, 198

pH 7.0) at 40 °C, in 2 mL Eppendorf tubes in a thermomixer (1000 rpm) in the dark, in 199

the absence or presence of 50 µM AscA. AnGOX converts glucose to gluconic acid and 200

produces H2O2. AnGOX is an FAD-dependent oxidase with high specificity for glucose 201

that belongs to the family of GMC oxidoreductases classified in the AA3 CAZy family 202

and carries out a two-electron reduction of O2 to produce H2O22–4

. Before product 203

quantification, cello-oligosaccharides were hydrolyzed by TfCel5A, yielding oxidized 204

products with a degree of polymerization of 2 and 3 [GlcGlc1A, (Glc)2Glc1A], summed 205

up to yield the concentration of oxidized sites. 206

Panel (b) shows the oxidative activity of ScLPMO10C shown in panel (a) as a function 207

of the H2O2 production ability of AnGOX. The plot shows a linear correlation between 208

the rate of the LPMO and GOX activity. The apparent stoichiometry is lower than unity, 209

which is partly due to underestimation of LPMO-generated oxidized products (only the 210

soluble fraction was analyzed) and likely also to incomplete utilization of H2O2 by the 211

LPMO. The activity of GOX on glucose (15 mM) was assayed by the standard Amplex 212

Red/HRP method for all the AnGOX concentrations employed in panel (a) but in reaction 213

mixtures lacking the LPMO, in sodium phosphate buffer (50 mM, pH 7.0) at 40 °C. The 214

reactions were directly carried out in a 96-well microtiter plate to allow online 215

monitoring of the Amplex Red oxidation product (i.e. resorufin) at 540 nm generated by 216

an excess of HRP (i.e. 0.183 µM). This method yielded a linear relationship (not shown) 217

between the AnGOX concentration and the H2O2 production rate. Error bars show ± s.d. 218

(n = 3) for y-values in panel (a) and for x and y-values in panel (b). 219


11

220 Supplementary Figure 8 Product profile (HPAEC-PAD) obtained after 221

degradation of cellulose by ScLPMO10C in reactions carried out in aerobic or 222

anaerobic conditions, in the presence or absence of initial exogenous H2O2 (100 µM). 223

Anaerobic or aerobic solutions of ScLPMO10C-Cu(II) (0.5 µM) and Avicel (10 g.L-1

) 224

prepared in sodium phosphate buffer (50 mM, pH 7.0) were incubated at 30 °C under 225

magnetic stirring, supplemented or not with 100 µM H2O2. Reactions were initiated by 226

the addition of AscA (1 mM). Each chromatogram is the average of 3 replicates and 227

shows the product profile after 30 min of incubation. The small peaks observed in the 228

anaerobic control reaction (black line) correspond to background signals present in the 229

substrate. Sampling at 60 or 90 min (not shown) led to identical chromatograms 230

indicating that the reaction was already over after 30 min. Note that the experimental 231

conditions of this experiment differ from the conditions used in Fig. 1d of the main 232

paper, where lower AscA concentrations were used. In the present experiment a relatively 233

high concentration of AscA (i.e. 1 mM) was used to make sure that, if O2 was present in 234

the reaction mix, LPMO activity would be detected. In such conditions, the absence of 235

oxidized cello-oligosaccharides in the negative control (black line) validates the protocol 236

employed to reach sufficient anaerobiosis (See online methods section). 237

238


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239 240

Supplementary Figure 9 Probing the origin of oxygen introduced into the 241

polysaccharide chain by ScLPMO10C using H218

O2. MALDI-TOF MS spectra of 242

products obtained after 4 min reaction in the presence of 100 µM H216

O2 or H218

O2, as 243

indicated, and 1 mM AscA. The spectrum shows the hexose cluster, showing sodium 244

adducts of the native (Nat) hexose, and the two forms of the oxidized hexose, the lactone 245

(Lac) and the aldonic acid (Ald). The spectra show that when using H218

O2, the 246

characteristic signals for sodium adducts of the aldonic acid form of an oxidized 247

cellohexaose (m/z 1029.7 & 1051.7) shifted by +2 Da. All reactions were carried out with 248

ScLPMO10C-Cu(II) (0.5 µM) and Avicel (10 g.L-1

) in sodium phosphate buffer (50 mM, 249

pH 7.0) at 40 °C under magnetic stirring. Abbreviations: DP, degree of polymerization; 250

Nat, native; Lac, oxidized, lactone form; Ald, oxidized, aldonic acid form. Nb: The small 251

peak observed at m/z = 1051.7 is due to the presence of small amounts of H216

O2 in the 252

H218

O2 solution and/or slow exchange of the aldonic acid with bulk solvent. The 253

experiment shown in this Figure was repeated several times with similar results; the 254

Figure shows a representative experiment. 255


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256 257

Supplementary Figure 10 Assessing the competition between H2O2 and O2 by 258

using low concentrations of H218

O2. ScLPMO10C (0.5 µM) was incubated in sodium 259

phosphate buffer (50 mM, pH 7.0) with Avicel (10 g.L-1

) at 40 °C under magnetic stirring 260

during 20 min before addition of a “priming” amount of AscA (10 µM) to initiate the 261

reaction. H218

O2 (25, 50 or 100 µM) was added to the reaction mixture just before AscA 262

addition. The graph is a zoom-out view of Fig 1e in the main article and shows MALDI-263

TOF MS spectra for the DP6 cluster, showing sodium adducts of the native (Nat), lactone 264

(Lac) and the aldonic acid (Ald) form. The graph shows the product profile obtained after 265

4 min reaction. Note that under the conditions used here the concentration of (non-266

labeled) 16

O2 in solution is in the range of 200-250 µM. Abbreviations: DP, degree of 267

polymerization; Nat, native; Lac, oxidized, lactone form; Ald, oxidized, aldonic acid 268

form. Nb: The small peak observed at m/z = 1051.7 is due to the presence of small 269

amounts of H216

O2 in the H218

O2 solution and/or slow exchange of the aldonic acid with 270

bulk solvent. The experiment shown in this Figure was repeated several times with 271

similar results; the Figure shows a representative experiment. 272


14

273 274

Supplementary Figure 11 Probing the origin of oxygen introduced into the 275

polysaccharide chain by the chitin-active AA10 CBP21 using H218

O2. CBP21 (0.5 276

µM) was incubated in sodium phosphate buffer (50 mM, pH 7.0) with β-chitin (10 g.L-1

) 277

at 40 °C under magnetic stirring during 20 min before addition of AscA (10 µM) to 278

initiate the reaction. When stated H216

O2 (purple line) or H218

O2 (orange line) (100 µM) 279

was added to the reaction mixture just before AscA addition. The graphs show MALDI-280

TOF MS spectra for the DP6 cluster for samples obtained after 60 minutes of incubation, 281

showing sodium adducts of the native (Nat), the lactone (Lac) and the aldonic acid (Ald) 282

form. Note that under the conditions used here the concentration of (non-labeled) 16

O2 in 283

solution is in the range of 200-250 µM. Abbreviations: DP, degree of polymerization; 284

Nat, native; Lac, oxidized, lactone form; Ald, oxidized, aldonic acid form. Nb: The small 285

peak observed at m/z = 1298 is due to the presence of small amounts of H216

O2 in the 286

H218

O2 solution and/or slow exchange of the aldonic acid with bulk solvent. The 287

experiment shown in this Figure was repeated several times with similar results; the 288

Figure shows a representative experiment. 289

290


15

291 292

Supplementary Figure 12 Probing the origin of the oxygen introduced into the 293

polysaccharide chain by the fungal cellulose-active PcLPMO9D using H218

O2. 294

PcLPMO9D (1 µM) was incubated in sodium phosphate buffer (50 mM, pH 7.0) with 295

PASC (9.5 g.L-1

) at 40 °C under magnetic stirring during 20 min before addition of AscA 296

(100 µM) to initiate the reaction. H216

O2 (purple line) or H218

O2 (orange line) (200 µM) 297

was added to the reaction mixture just before AscA addition. The graph shows MALDI-298

TOF MS spectra for the DP6 cluster, showing sodium adducts of the native (Nat), lactone 299

(Lac) and aldonic acid (Ald) form. The graph shows the product profile obtained after 15 300

min reaction. Note that under the conditions used here the concentration of (non-labeled) 301 16

O2 in solution is in the range of 200-250 µM. Abbreviations: DP, degree of 302

polymerization; Nat, native; Lac, oxidized, lactone form; Ald, oxidized, aldonic acid 303

form. Nb: The small peak observed at m/z = 1051.6 is due to the presence of small 304

amounts of H216

O2 in the H218

O2 solution and/or slow exchange of the aldonic acid with 305

bulk solvent. The experiment shown in this Figure was repeated several times with 306

similar results; the Figure shows a representative experiment. 307

308


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309 310

Supplementary Figure 13 Inhibition of ScLPMO10C-catalyzed degradation of 311

Avicel by competition for H2O2 with the HRP/Amplex Red system. Panels (a) and (c) 312

show time-courses for the release of aldonic acids from Avicel (10 g.L-1

) by 313

ScLPMO10C (0.5 µM) in reactions containing varying amount of a H2O2-consuming 314

enzyme (HRP), as explained in detail below. Panels (b) and (d) show the relative activity 315

of ScLPMO10C (derived from panels (a) and (c), respectively) as a function of the molar 316

ratio between HRP and the LPMO. Before product quantification, cello-oligosaccharides 317

were hydrolyzed by TfCel5A, yielding oxidized products with a degree of polymerization 318

of 2 and 3 [GlcGlc1A, (Glc)2Glc1A], summed up to yield the concentration of oxidized 319

sites. Error bars show ± s.d. (n = 3). 320

Reactions (200 µL total volume) were carried out in sodium phosphate buffer (50 321

mM, pH 7.0) at 40 °C, in 2 mL Eppendorf tubes in a thermomixer (1000 rpm), and 322

started by adding, in panel (a), AscA (1 mM, ‘+’ symbol) or, in panel (c), MtCDH (1 323

µM). Lactose (15 mM) was present as a substrate in all reactions containing MtCDH. In 324

panel (c), the activity of MtCDH is also shown and expressed as µM of oxidized lactose 325

(secondary y-axis), which was monitored simultaneously with the other aldonic acids. 326

The data presented in panel (a) and (c) show the effect of HRP (0-100 µg solid 327

powder.mL-1

) on LPMO activity. The acceptor chosen for the HRP was Amplex red (200 328

µM final concentration; present in all reactions indicated by a ‘+’ symbol, or replaced by 329

DMSO if not present). 330

Monitoring of remaining AscA (data not shown) showed that for all conditions 331

shown in panel (a) at least 0.8 mM AscA remained after 60 min. The data for lactose 332

oxidation in panel (c) show that MtCDH was active in all conditions used. Dissolved O2 333

was also monitored and found to be stable at 170 - 200 µM for all conditions. 334


17

335 336

Supplementary Figure 14 Putative reaction mechanisms for polysaccharide 337

oxidation by LPMOs using H2O2 (left side) or O2 (right side) as co-substrate. In all 338

cases the entry point in the catalytic cycle is the single electron reductive activation of 339

LPMO-Cu(II) to LPMO-Cu(I). From this point several scenarios can be envisioned: H2O2 340

reacts with the Cu(I) center leading to the production of a hydroxyl radical via homolytic 341

bond cleavage (pathway I) or via a base-assisted mechanism (pathway II). In the base-342

assisted mechanism, concomitant proton abstraction from H2O2 by the base and reduction 343

of H2O2 by Cu(I) leads to an intermediate with an elongated O-O bond, displayed with 344

three electrons and a dotted line for illustrative purposes. The proton that is held by the 345

putative base can react either with the copper-bound oxygen atom (pathway II-a, grey) 346

or with the leaving hydroxide group (pathway II-b, magenta), which leads to elimination 347

of a water molecule and formation of a copper-oxyl intermediate. Pathways (I) and (II-a) 348

both lead to the formation of a Cu(II)-hydroxide intermediate and a hydroxyl radical. 349

This hydroxyl radical catalyzes HAA either from the Cu(II)-hydroxide (haa1) or from the 350

substrate (haa1’). The former scenario leads to a Cu(II)-oxyl intermediate that can 351

catalyze HAA on the substrate (haa2). In both cases (haa1+haa2 or haa1´), a water 352

molecule is eliminated and a substrate radical (R) and a common Cu(II)-OH 353

intermediate are generated. The Cu(II)-associated hydroxide merges with the substrate 354

radical through a rebound mechanism, leading to hydroxylation of the substrate and 355

regeneration of the Cu(I) center, which can enter a new catalytic cycle. The pathway 356

involving haa2 is similar to the last part of the copper-oxyl, oxygen-rebound mechanism 357

(in which the first step was reductive activation of O2, see below pathway III-c) 358


18

proposed by Kim et al. on the basis of QM calculations5. Also, compared to haa1´, the 359

haa1 pathway appears most likely since the generation of a seemingly free hydroxyl 360

radical in the haa1´ pathway is hardly compatible with the high regioselectivity displayed 361

by the LPMO. In the H2O2-based mechanisms no exogenous source of protons or 362

electrons is required once the priming reduction of the LPMO has been achieved, as 363

opposed to O2-based mechanisms. 364

In the O2-based mechanisms (right side of the figure), the LPMO-Cu(II)-365

superoxide intermediate resulting from the reaction between the LPMO-Cu(I) and O2 366

(pathway III6) can take several routes. In the absence of substrate (pathway III-a) 367

superoxide is released 6, leading to the regeneration of the initial LPMO-Cu(II) state and 368

to H2O2 production from superoxide disproportionation. In the presence of substrate, 369

different routes are conceivable5,7–9

and all require the recruitment of 2 electrons and 2 370

protons per catalytic cycle. One of the routes (pathway III-c) involves formation of a 371

copper-oxyl intermediate5 and is therefore connected to the H2O2-pathways in this 372

drawing. 373

The base-assisted variant of the H2O2 reaction scheme (pathway II) is a 374

possibility that has to be considered since examination of available crystallographic 375

structures reveals a structurally conserved base residue (glutamate) in all strictly chitin-376

active and cellulose-active C1-specific LPMO10s (e.g. E217 in ScLPMO10C and E60 in 377

CBP21) as well as in chitin-active LPMO11. However, a glutamine is found at the 378

equivalent position in LPMO10s with C1/C4 mixed activity (e.g. Q217 in ScLPMO10B) 379

and in LPMO9s (e.g. Q176 in PcLPMO9D). 380

The present study proposes and demonstrates that the LPMO takes the H2O2-381

based route and multi-disciplinary approaches will have to be developed to unravel the 382

mechanistic details underlying this pathway. Importantly, the H2O2-pathway described 383

here for LPMOs should not be confused with a “peroxide shunt” pathway in the sense 384

that H2O2 is not involved in a bypass reaction but is a strictly required co-substrate for 385

catalysis to occur, as shown by the variety of competition experiments (See Fig 1e&f in 386

the main article and Supplementary Figs 9, 10&13). The present data also show that 387

after an essential priming reduction, stable reaction kinetics with multiple turnovers are 388

achieved at low (low M range) H2O2 concentrations, in contrast with the rather artificial, 389

slow peroxide shunts described in the literature that require high concentrations of H2O2 390

(10-100 mM)10,11

and that tend to lead to unstable reactions with a limited number of 391

turnovers. 392

393


19

394 395

Supplementary Figure 15 Study of ScLPMO10C inactivation by the Chl+AscA 396

system in the dark or in the light. The experiment consisted of two phases, namely a 397

pretreatment phase followed by an activity test phase. During the first phase 398

ScLPMO10C (0.5 µM) was incubated in sodium phosphate buffer (50 mM, pH 6.0) at 40 399

°C under magnetic stirring in presence of Chl (500 µM) and AscA (1 mM). Conditions 400

varied in terms of light exposure [visible light, 25% Imax (eq. 42 W.cm-2

), or dark], 401

presence or absence of Avicel (10 g.L-1

) and presence or absence of EDTA (0.5 mM; to 402

chelate metals in the substrate; this EDTA concentration is not sufficient to chelate the 403

copper from the LPMO). These conditions were used because they lead to high activity 404

and fast enzyme inactivation (See Supplementary Fig. 2). After 2 h of incubation, half 405

of the pre-treated sample (250 µL) was directly transferred to 245 µL of a pre-incubated 406

(20 min, 40 °C) suspension of Avicel (10 g.L-1

) in sodium phosphate buffer (50 mM, pH 407

6.0). To secure initiation of the second phase reaction AscA was added (5 µL of a 100 408

mM solution, yielding 1 mM final concentration). The second phase reaction mixtures 409

were incubated in a thermomixer (40 °C, 850 rpm) for up to 8 h, after which the release 410

of oxidized products was analyzed. For the second phase, two control reactions were set 411

up containing ScLPMO10C (0.25 µM), in the presence (0.25 mM) or absence of EDTA, 412

incubated with Avicel (10 g.L-1

) in sodium phosphate buffer (50 mM, pH 6.0) in a 413

thermomixer (40 °C, 850 rpm), and these reactions were initiated by addition of AscA (1 414

mM). The data shown in this Figure are from a single experiment. 415

416


20

417 418

Supplementary Figure 16 Semi-quantitative distribution of selected 419

modifications observed in samples of pretreated LPMO. The histograms show for 420

each sample the abundance of native (m/z change 0.00) or modified peptides bearing (a) 421

the catalytic histidines (H35 and H144), (b) tryptophanes W123, W141 and W210, (c) 422

tyrosines Y111, Y116 and Y138 and (d) phenylalanines F113 and F207. This semi-423


21

quantitative distribution analysis has been done for treated enzyme samples (sample #1 to 424

4; see below for description of treatment) and should be compared to the analysis for an 425

untreated control (#5, black bars). In the treated samples, the catalytic histidines are 426

heavily modified, with the proportion of modified peptides being larger that the 427

proportion of native peptides, whereas the untreated sample #5 displays mainly native 428

peptides. The main modifications observed on histidines are oxidations (oxo-His), 429

followed by ring opening and further degradation down to aspartate (m/z = -22) or 430

asparagine (m/z = -23), as described by Uchida et al.12

. Regarding tryptophans, a series of 431

oxidative modifications, the mechanism of which is described in the literature13

, are 432

observed. However, these modifications are also quite abundant in the untreated protein. 433

Thus, these modifications likely reflect in part chemical artifacts due to sample 434

processing13

. For tyrosines and phenylalanine, hydroxylation events were detected, but 435

these were not very abundant and not much more prominent in the pretreated samples 436

relative to the control sample. The modifications that were selected for further 437

quantitative analysis are highlighted by a green arrow. The data shown in this Figure are 438

from a single experiment. 439

Pre-treatment conditions were as follows: ScLPMO10C (1 µM, eq. 17.3 µg) was 440

exposed to [Chl/light+AscA] (#1 and #2) or to AscA (#3 and #4), in the presence (#1 and 441

#3) or absence (#2 and #4) of Avicel (10 g.L-1

). A control experiment (#5) was carried 442

out in the absence of substrate and electron source. All reactions were incubated during 2 443

hours in sodium phosphate buffer (50 mM, pH 6.0), under magnetic stirring at 40 °C. 500 444

µM of Chl, 1 mM of AscA and a light intensity of 25% Imax (eq. 42 W.cm-2

) were 445

employed, as indicated. These conditions lead to rapid inactivation of the enzyme, as 446

illustrated by Supplementary Fig. 2. 447

448


22

449 450

Supplementary Figure 17 Identification of residues modified during 451

LPMO inactivation. (a) Ratio (R) between the fraction of modified peptides in sample x 452

(#1 - #4) and the fraction of modified peptides in sample #5 for peptides modified at 453

position y (indicated above the chart). (see Materials and methods section, R = 1 for 454

sample #5). (b) Significance factor for modification of the indicated residues in treated 455

samples of ScLPMO10C. The significance factor equals R times the frequency of 456

modification; see Materials and methods section). From this analysis, residues 457

considered as significantly affected are the catalytic histidines, H35 and H144, N140, 458

and, to a lesser extent, Y111, Y138 and W141 (See Supplementary Fig. 18 for the 459

position of these residues in sequence and structure). For pretreatment conditions, see the 460

legend of Supplementary Fig. 16. The data shown in this Figure are from a single 461

experiment. 462

463

464


23

465 Supplementary Figure 18 Location of oxidative modifications in ScLPMO10C. (a, 466

identical to Fig. 2 in the main text) Mapping of modified residues on the structure of the 467

catalytic domain of ScLPMO10C (PDB 4OY714

) reveals that oxidation occurs in and near 468

the active site, predominantly on the catalytic histidines, H35 (= the N-terminus) and 469

H144. The color code highlights the degree of oxidation: high (red), middle (orange) and 470

low (yellow). For aromatic residues shown as grey sticks no modification was detected 471

(See Supplementary Fig. 16-17). (b) The mature protein, used for the study, does not 472

contain the signal peptide (highlighted in green). The protein is composed of the LPMO 473

domain (blue) and a CBM (yellow), connected by a linker (magenta). Modifications 474

considered as significant (big or small red star; see Supplementary Fig. 17) occur in the 475

LPMO domain only, whereas the linker and the CBM are not affected. The sequence 476

coverage of all the samples analyzed in this study was in between 78 % and 90% (The 477

sequence underlined in red represents a non-covered region). 478


24

479 480

Supplementary Figure 19 Inactivation of ScLPMO10C by AscA/H2O2 and 481

the protective role of the substrate. The experiment consisted of two phases, namely a 482

pretreatment phase followed by an activity test phase. During the first phase 483

ScLPMO10C (1 µM) was incubated in sodium phosphate buffer (50 mM, pH 7.0) at 40 484

°C under magnetic stirring, in the presence (10 g.L-1

) or absence of Avicel during 20 min 485

before addition of either AscA (1mM)/H2O2 (100 µM) or simply water and further 486

incubation for 20 min. This first phase is the so-called pre-treatment phase. Then, half of 487

the pre-treated sample (250 µL) was directly transferred to 245 µL of a pre-incubated (20 488

min, 40 °C) suspension of Avicel (10 g.L-1

) prepared in sodium phosphate buffer (50 489

mM, pH 7.0). To ensure activity, fresh AscA was also added (5 µL of a 100 mM solution, 490

giving a final added concentration of 1 mM). The reactions were incubated in a 491

thermomixer (40 °C, 850 rpm) for up to 20 h. In control samples ScLPMO10C (1 µM) 492

was subjected to the same protocol as treated samples (in presence or absence of Avicel) 493

but without AscA and H2O2. The release of oxidized products during this second phase 494

was monitored and is shown in panel (a). Before product quantification, 495

celloligosaccharides were hydrolyzed by TfCel5A, yielding oxidized products with a 496

degree of polymerization of 2 and 3 [GlcGlc1A, (Glc)2Glc1A], summed up to yield the 497


25

concentration of oxidized sites. The experiment depicted in the Figure was performed to 498

validate the protocol of LPMO inactivation/substrate protection employed for the analysis 499

of LPMO self-oxidation by HPLC-MS/MS (See panel b). The error bars show s.d. (n 500

=2). (b) Impact of substrate on the ratio of modified/native peptides bearing H35, N140, 501

W141 or H144 after a short incubation (i.e. a less drastic treatment compared to 502

Supplementary Fig. 15-18). ScLPMO10C (1 µM) was pre-treated by 20 min incubation 503

in sodium phosphate buffer (50 mM, pH 7.0) at 40 °C under magnetic stirring, in the 504

presence (10 g.L-1

) or absence of Avicel and addition of either AscA (1mM)/H2O2 (100 505

µM) or simply water (control reaction), as indicated. 506

507


26

3. Full abbreviations list 508 509 Ald: aldonic acid form of oxidized product 510

AnGOX: Glucose oxidase from Aspergillus niger 511

AscA: ascorbic acid 512

CBM: carbohydrate binding module 513

CBP21: LPMO10 from Serratia marcescens 514

MtCDH: cellobiose dehydrogenase from Myriococcum thermophilum 515

Chl: chlorophyllin 516

GH: Glycoside hydrolases 517

GMC: Glucose-methanol-choline oxidoreductase 518

HAA: hydrogen atom abstraction 519

HRP: Horseradish peroxidase 520

Lac: lactone form of oxidized product 521

LPMO: lytic polysaccharide monooxygenases 522

Nat: native form of oligosaccharide 523

PcLPMO9D: LPMO9D from Phanerochaete chrysosporium K-3 524

ScLPMO10B and ScLPMO10C: LPMO10B and 10C from Streptomyces coelicolor 525

TfCel5A: Cel5A from Thermobifida fusca 526

XTH: xanthine 527

XOD: xanthine oxidase 528

529


27

4. Supplementary references 530

531 1. Cannella, D. et al. Light-driven oxidation of polysaccharides by photosynthetic pigments and a 532

metalloenzyme. Nat. Comm. 7, 11134 (2016). 533 2. Pazur, J. H. & Kleppe, K. The oxidation of glucose and related compounds by glucose oxidase 534

from Aspergillus niger. Biochemsitry 3, 578–583 (1964). 535 3. Leskovac, V., Trivić, S., Wohlfahrt, G., Kandrač, J. & Peričin, D. Glucose oxidase from 536

Aspergillus niger: the mechanism of action with molecular oxygen, quinones, and one-electron 537 acceptors. Int. J. Biochem. Cell Biol. 37, 731–750 (2005). 538

4. Vuong, T. V., Foumani, M., MacCormick, B., Kwan, R. & Master, E. R. Direct comparison of 539 gluco-oligosaccharide oxidase variants and glucose oxidase: substrate range and H2O2 stability. Sci. 540 Rep. 6, 1–9 (2016). 541

5. Kim, S., Ståhlberg, J., Sandgren, M., Paton, R. S. & Beckham, G. T. Quantum mechanical 542 calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound 543 mechanism. Proc. Natl. Acad. Sci. U. S. A. 111, 149–154 (2014). 544

6. Kjaergaard, C. H. et al. Spectroscopic and computational insight into the activation of O2 by the 545 mononuclear Cu center in polysaccharide monooxygenases. Proc. Natl. Acad. Sci. U. S. A. 111, 546 8797–8802 (2014). 547

7. Walton, P. H. & Davies, G. J. On the catalytic mechanisms of lytic polysaccharide 548 monooxygenases. Curr. Opin. Chem. Biol. 31, 1–13 (2016). 549

8. Beeson, W. T., Vu, V. V., Span, E. A., Phillips, C. M. & Marletta, M. A. Cellulose degradation by 550 polysaccharide monooxygenases. Annu. Rev. Biochem. 84, 923–946 (2015). 551

9. Lee, J. Y. & Karlin, K. D. Elaboration of copper-oxygen mediated CH activation chemistry in 552 consideration of future fuel and feedstock generation. Curr. Opin. Chem. Biol. 25, 184–193 (2015). 553

10. Cirino, P. C. & Arnold, F. H. A self-sufficient peroxide-driven hydroxylation biocatalyst. Angew. 554 Chemie - Int. Ed. 42, 3299–3301 (2003). 555

11. Hrycay, E. G. & Bandiera, S. M. Monooxygenase, peroxidase and peroxygenase properties and 556 reaction mechanisms of cytochrome P450 enzymes. Adv. Exp. Med. Biol. 851, 1–61 (2012). 557

12. Uchida, K. Ascorbate-mediated specific oxidation of the imidazole in a histidine derivative. Bioorg. 558 Chem. 343, 330–343 (1989). 559

13. Perdivara, I., Deterding, L. J., Przybylski, M. & Tomer, K. B. Mass spectrometric identification of 560 oxidative modifications of tryptophan residues in proteins: Chemical artifact or post-translational 561 modification? J. Am. Soc. Mass Spectrom. 21, 1114–1117 (2010). 562

14. Forsberg, Z. et al. Structural and functional characterization of a conserved pair of bacterial 563 cellulose-oxidizing lytic polysaccharide monooxygenases. Proc. Natl. Acad. Sci. U. S. A. 111, 564 8446–8451 (2014). 565

566


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