Bilal Alessia Munzone

El Kerdi

Mathieu Fanuel

Hélène Rogniaux

David Ropartz

Marius Réglier

Antoine Royant

A Jalila Simaan

Christophe Decroos
email: christophe.decroos@univ-amu.fr

Bilal El Kerdi

Characterization of a bacterial copper-dependent lytic polysaccharide monooxygenase with an unusual second coordination sphere

Keywords: Lytic polysaccharide monooxygenase, copper metalloenzyme, chitin oxidation, Photorhabdus luminescens, X-ray crystallography carbohydrate-active enzyme, CID, collision-induced dissociation, DP, degree of polymerization, EPR, electron paramagnetic resonance, ESI, electrospray ionization, IPTG, isopropyl-β-D-thiogalactopyranoside, LB, lysogeny broth, LPMO, lytic polysaccharide monooxygenase, MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight, MS, mass spectrometry, MWCO, molecular weight cutoff, PlAA10, LPMO from Photorhabdus luminescens, rmsd, root-mean-square deviation, TSB, tryptic soy broth

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Introduction

Cellulose and chitin are two most abundant biopolymers in nature, constituted of tightly packed polysaccharide chains of β- [START_REF] Cosgrove | Growth of the plant cell wall[END_REF][START_REF] Vaaje-Kolstad | The noncatalytic chitin-binding protein CBP21 from Serratia marcescens is essential for chitin degradation[END_REF]-linked glucose and N-acetylglucosamine units, respectively. Thanks to their crystallinity conferring strength and recalcitrance, these biopolymers are found as major structural components of plant cell wall for cellulose [START_REF] Cosgrove | Growth of the plant cell wall[END_REF], and of the exoskeleton of arthropods such as crustaceans and insects, as well as of the fungal and yeast cell walls for chitin [START_REF] Cohen | Chitin synthesis and inhibition: a revisit[END_REF]. Synergistic endo-and exo-acting glycoside hydrolases -including cellulases and chitinases -secreted by cellulose-and chitin degrading microorganisms (bacteria and fungi) were long considered to be the only enzymatic partners involved in recalcitrant polysaccharide breakdown. However, Reese et al. suggested that an additional non-hydrolytic factor could reduce cellulose recalcitrance prior to enzymatic hydrolysis by fungal cellulases [START_REF] Reese | The biological degradation of soluble cellulose derivatives and its relationship to the mechanism of cellulose hydrolysis[END_REF]. This extra component remained elusive until a decade ago with the discovery of new proteins able to enhance the hydrolytic activity of chitinases and cellulases [START_REF] Vaaje-Kolstad | The noncatalytic chitin-binding protein CBP21 from Serratia marcescens is essential for chitin degradation[END_REF][START_REF] Harris | Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family[END_REF]. Notably, these enzymes called lytic polysaccharide monooxygenases (LPMOs) were later found to introduce chain breaks into the polysaccharide via an oxidative mechanism [START_REF] Vaaje-Kolstad | An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[END_REF] relying on a mononuclear copper ion in their active site [START_REF] Quinlan | Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components[END_REF][START_REF] Phillips | Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa[END_REF]. This disrupts locally the substrate crystallinity on its surface, which facilitates the action of glycoside hydrolases working in concert with LPMOs and thus boosts the degradation of recalcitrant polysaccharides. LPMOs are now classified as auxiliary activities (AA) in the carbohydrate-active enzymes database (CAZy database, http://www.cazy.org) due to their redox function [START_REF] Lombard | The carbohydrate-active enzymes database (CAZy) in 2013[END_REF][START_REF] Levasseur | Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes[END_REF], and are divided into seven families: AA9 (formerly GH61) [START_REF] Quinlan | Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components[END_REF], AA10 (formerly CBM33) [START_REF] Vaaje-Kolstad | An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[END_REF], AA11 [START_REF] Hemsworth | Discovery and characterization of a new family of lytic polysaccharide monooxygenases[END_REF], AA13 [START_REF] Vu | A family of starchactive polysaccharide monooxygenases[END_REF][START_REF] Lo Leggio | Structure and boosting activity of a starch-degrading polysaccharide monooxygenase[END_REF], AA14 [START_REF] Couturier | Lytic xylan oxidases from wood-decay fungi unlock biomass degradation[END_REF], AA15 [START_REF] Sabbadin | An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion[END_REF], and AA16 [START_REF] Filiatrault-Chastel | AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes[END_REF].

LPMOs catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides (Figure 1a). Notably, they hydroxylate the strong C-H bond either at position C1 or C4. The resulting unstable intermediate further decomposes leading to different products. C1 oxidation produces aldonolactones that are hydrolyzed to their corresponding aldonic acids. C4 oxidation generates ketoaldose products that are in equilibrium with their hydrated gem-diol forms.

Generally, chitin-active LPMOs regiospecifically oxidize their substrate at position C1 [START_REF] Vaaje-Kolstad | An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[END_REF][START_REF] Hemsworth | Discovery and characterization of a new family of lytic polysaccharide monooxygenases[END_REF][START_REF] Sabbadin | An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion[END_REF][START_REF] Forsberg | Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases[END_REF][START_REF] Nakagawa | A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting aand b-chitin[END_REF][START_REF] Gregory | Activity, stability and 3-D structure of the Cu(II) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens[END_REF]. On the contrary, some LPMOs active on cellulose were found to exclusively oxidize at position C1 or C4, while others can generate oxidation product originating from both C1 and C4 oxidation [START_REF] Phillips | Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa[END_REF][START_REF] Filiatrault-Chastel | AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes[END_REF][START_REF] Forsberg | Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases[END_REF][START_REF] Forsberg | Cleavage of cellulose by a CBM33 protein[END_REF][START_REF] Beeson | Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases[END_REF][START_REF] Forsberg | Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases[END_REF][START_REF] Gardner | Systems biology defines the biological significance of a redox-active proteins during cellulose degradation in an aerobic bacterium[END_REF][START_REF] Ghatge | Multifunctional cellulolytic auxiliary activity protein HcAA10-2 from Hahella chejuensis enhances enzymatic hydrolysis of crystalline cellulose[END_REF][START_REF] Forsberg | Structural determinants of bacterial lytic polysaccharide monooxygenase functionality[END_REF][START_REF] Fowler | Discovery, activity and characterization of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae[END_REF]. As previously mentioned, LPMOs reactivity depends on a catalytically obligatory copper ion at their active site. The Cu(II) resting state of LPMOs needs to be reduced to Cu(I) to initiate catalysis. Electrons are supplied by either proteic partners or by small molecule reductants [START_REF] Bissaro | Oxidoreductases and reactive oxygen species in conversion of lignocellulosic biomass[END_REF]. Upon Cu(II) reduction to Cu(I), LPMOs can activate their co-substrate that can be either dioxygen [START_REF] Vaaje-Kolstad | An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[END_REF][START_REF] Quinlan | Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components[END_REF][START_REF] Beeson | Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases[END_REF] or hydrogen peroxide [START_REF] Bissaro | Oxidoreductases and reactive oxygen species in conversion of lignocellulosic biomass[END_REF][START_REF] Bissaro | Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2[END_REF]. While the active species remains to be characterized, theoretical calculations identified a Cu(II)-oxyl as the intermediate responsible for substrate hydroxylation via hydrogen abstraction and oxygenrebound mechanism [START_REF] Wang | QM/MM studies into the H2O2-dependent activity of lytic polysaccharide monooxygenases: evidence for the formation of a caged hydroxyl radical intermediate[END_REF][START_REF] Hedegård | Molecular mechanism of lytic polysaccharide monooxygenases[END_REF]. LPMOs all adopt a common distorted immunoglobulin-like β-sandwich fold, although they share very low sequence identity between different families [START_REF] Vaaje-Kolstad | Structural diversity of lytic polysaccharide monooxygenases[END_REF]. They exhibit a relatively flat surface where the crystalline substrate binds [START_REF] Aachmann | NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions[END_REF]. The copper active site of LPMOs is located on this surface so that it is exposed to the substrate when bound. Two strictly conserved histidine residues coordinate the Cu(II) ion in a unique motif referred to as the histidine brace (Figures 1b and1c), in which the N-terminal histidine serves as bidentate ligand (coordination by both the N-terminal amine and the imidazole side chain Nδ1) [START_REF] Quinlan | Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components[END_REF][START_REF] Vaaje-Kolstad | Structural diversity of lytic polysaccharide monooxygenases[END_REF]. Two water molecules complete the Cu(II) first coordination sphere (Figure 1b). Due to X-ray photoreduction, most of the LPMOs structures are in the reduced Cu(I) state featuring a T-shaped geometry (no water molecule coordinated, Figure 1c) [START_REF] Hemsworth | The copper active site of CBM33 polysaccharide oxygenases[END_REF][START_REF] Gudmundsson | Structural and electronic snapshots during the transition from a Cu(II) to Cu(I) metal center of lytic polysaccharide monooxygenase by X-ray photoreduction[END_REF]. In fungal LPMOs, the Nε2 atom of the N-terminal histidine is posttranslationally methylated [START_REF] Quinlan | Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components[END_REF][START_REF] Lo Leggio | Structure and boosting activity of a starch-degrading polysaccharide monooxygenase[END_REF][START_REF] Vaaje-Kolstad | Structural diversity of lytic polysaccharide monooxygenases[END_REF]. The histidine brace motif is fairly rare as it has only been observed so far in a handful of copper-metalloproteins besides LPMOs, including the PmoB subunit of the copper-dependent particulate methane monooxygenase [START_REF] Lieberman | Crystal structure of a membrane-bound metalloenzyme that catalyses the biological oxidation of methane[END_REF][START_REF] Cao | Quantum refinement does not support dinuclear copper sites in crystal structures of particulate methane monooxygenases[END_REF], and the bacterial copper-transport proteins CopC [START_REF] Zhang | Intermolecular transfer of copper ions from the CopC protein of Pseudomonas syringae. Crystal structures of fully loaded Cu I Cu II forms[END_REF][START_REF] Lawton | The CopC family: structural and bioinformatic insights into a diverse group of periplasmic copper binding proteins[END_REF] and PmoF [START_REF] Fisher | PCuAC domains from methane-oxidizing bacteria use a histidine brace to bind copper[END_REF]. In LPMOs, a conserved tyrosine or phenylalanine residue is found in axial position of the copper coordination sphere [START_REF] Vaaje-Kolstad | Structural diversity of lytic polysaccharide monooxygenases[END_REF]. Interestingly, a highly conserved second coordination sphere alanine residue in AA10 LPMOs (mostly bacterial) was tentatively hypothesized to provide some steric hindrance close to the solvent-exposed axial position of the metal center and therefore orient the binding and reactivity of dioxygen or hydrogen peroxide in the vacant equatorial position of the Cu(I) [START_REF] Hemsworth | The copper active site of CBM33 polysaccharide oxygenases[END_REF].

Apart from their role in polysaccharidic biomass breakdown, AA10 LPMOs are also involved in several physiological processes including endosymbiosis (host-microbe interactions) and pathogenicity (virulence factors) [START_REF] Agostoni | Physiological and molecular understanding of bacterial polysaccharide monooxygenases[END_REF]. Photorhabdus luminescens is a Gramnegative broad-spectrum insect pathogenic bacterium that lives in symbiosis with the Heterorhabditis bacteriophora nematode. Photorhabdus luminescens is released in the insect larva hemocoel upon nematode invasion. The insect is rapidly killed after the production of toxins and virulence factors by the entomopathogenic bacteria. The insect cadaver is further bioconverted by enzymes secreted by the bacteria, supporting the growth of both the nematodes and Photorhabdus luminescens. Genes encoding for secreted enzymes involved in host chitin degradation (several chitinases and a single LPMO) were identified in Photorhabdus luminescens [START_REF] Duchaud | The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens[END_REF], as expected for insect pathogens [START_REF] Agostoni | Physiological and molecular understanding of bacterial polysaccharide monooxygenases[END_REF][START_REF] Nielsen-Leroux | How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts[END_REF][START_REF] Garcia-Gonzalez | Paenibacillus larvae chitin-degrading protein PlCBP49 is a key virulence factor in American foulbrood of honey bees[END_REF]. In this work, we characterized the single LPMO from Photorhabdus luminescens, referred to as PlAA10. Sequence analysis revealed that PlAA10 lacks the conserved alanine residue found in the active site of AA10 LPMOs (Figure 1b). Instead, this second coordination sphere residue is substituted by a bulkier isoleucine in PlAA10. This study reports the biophysical, biochemical, and structural characterization of this AA10 LPMO featuring an unusual second coordination sphere. Overall, this study provides new insights into the diversity observed among bacterial LPMOs.

Results and Discussion

The putative chitin-active LPMO PlAA10 harbors an isoleucine in place of the conserved

active-site alanine residue

The genome from the entomopathogenic bacteria Photorhabdus luminescens contains over 70 genes encoding carbohydrate-active enzymes (CAZymes). Among this CAZymes, several chitinases and a single LPMO are predicted to be essential for insect larva chitin degradation during invasion, and later during bioconversion of its cadaver [START_REF] Duchaud | The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens[END_REF][START_REF] Nielsen-Leroux | How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts[END_REF][START_REF] Garcia-Gonzalez | Paenibacillus larvae chitin-degrading protein PlCBP49 is a key virulence factor in American foulbrood of honey bees[END_REF]. Accordingly, chitinase activity has already been reported in culture supernatants of Photorhabdus luminescens and other related insect pathogenic bacteria [START_REF] Chen | Chitinase activity of Xenorhabdus and Photorhabdus species, bacterial associates of entomopathogenic nematodes[END_REF]. Nevertheless, no chitinolytic enzyme (chitinase or LPMO) from Photorhabdus luminescens has been characterized so far. The single LPMO from Photorhabdus luminescens, referred to as PlAA10, is predicted to be secreted as a singledomain enzyme (catalytic domain: residues 26-199) after cleavage of its signal peptide (residues 1-25). From the phylogenetic analysis we performed (Figure 2a), PlAA10 is implied to catalyze the oxidative cleavage of the glycosidic bond of chitin as it clusters with other characterized chitin-active LPMOs. In particular, PlAA10 exhibits 52% sequence identity with the well-characterized chitin-active LPMO SmAA10 (also known as CBP21) from the chitinolytic bacterium Serratia marcescens [START_REF] Vaaje-Kolstad | An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[END_REF][START_REF] Aachmann | NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions[END_REF][START_REF] Vaaje-Kolstad | Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21[END_REF][START_REF] Bissaro | How a lytic polysaccharide monooxygenase binds crystalline chitin[END_REF]. The two strictly conserved histidine residues required for copper binding are present in PlAA10 -H26 and H115 defining the histidine brace -as well as the conserved second coordination sphere axial phenylalanine F188 (Figure 1b). Interestingly, multiple sequence alignment revealed that no conserved alanine is found within the active site of PlAA10, but an isoleucine residue instead (I113) (Figure 2b). To preclude any possible previous sequencing error, a 1 kb fragment including the gene coding for PlAA10 (plu2352) was amplified from Photorhabdus luminescens genomic DNA and further sequenced, which confirmed the presence of an unusual alanine-to-isoleucine substitution in PlAA10. To date, all characterized chitin-active AA10 LPMOs possess the conserved active-site alanine residue. We were therefore interested in studying the structural and functional properties of this enzyme. [START_REF] Nakagawa | A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting aand b-chitin[END_REF]. Characterized members of subclades IV and VII are active on cellulose with a C1regiospecificity of oxidation. Cellulose oxidation at both position C1 and C4 were reported for Phylogenetic analyses have shown that cellulose-active AA10 LPMOs derive from chitinactive AA10 LPMOs. It has been proposed that the chitin activity of subclade V members could be a residual ancestral activity [START_REF] Book | Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases[END_REF]. So far, no member of subclade VI has been biochemically characterized. Nevertheless, the membrane-bound ScAA10D from Streptomyces coelicolor A3(2) was shown to bind crystalline cellulose [START_REF] Walter | Characteristics of the surface-located carbohydratebinding protein CbpC from Streptomyces coelicolor A3(2)[END_REF]. Some biochemically (m) and / or structurally (q) characterized AA10 LPMOs are highlighted. The origin and GenBank accession number of putative and characterized bacterial LPMOs included in this unrooted phylogenetic tree are given in Table S1. The multiple sequence alignment was generated with Clustal Omega and the unrooted phylogenetic tree drawn with Interactive Tree of Life (iTOL).

J d A A 1 0 B Sa lA A1 0C S fA A 1 0 C M a A A 1 0 B X c A A 1 0 B Sc AA 10 C SalA A10B SfAA 10B ScAA 10D M a A A 1 0 C H cA A1 0A ScAA 10 Sl AA10 S c A A 1 0 B S c A A 1 0 S fA A 1 0 L h A A 1 0 Sa lA A1 0A T f A A 1 0 B Sc AA 10 A M a A A 1 0 D X c A A 1 0 A M a A A 1 0 S fA A 1 0 A c A A 1 0 P l MaA A10A S c A A 1 0 H c A A
(b) Multiple sequence alignment of PlAA10 with previously characterized chitin-active AA10

LPMOs featuring the strictly conserved second histidine active site (*) and the conserved second coordination sphere alanine residue observed for all characterized chitin-active AA10 (p). The multiple sequence alignment was generated with Clustal Omega and visualized with Jalview.

PlAA10 binds copper similarly to other chitin-active LPMOs

In their native hosts, LPMOs are produced as secreted enzymes after cleavage of their signal peptide. This leads to a mature protein with a N-terminal histidine required for Cu(II)-binding (histidine brace) and therefore catalysis. Bacterial LPMOs are usually overexpressed in the periplasm of Escherichia coli [START_REF] Hemsworth | The copper active site of CBM33 polysaccharide oxygenases[END_REF][START_REF] Vaaje-Kolstad | Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21[END_REF][START_REF] Vaaje-Kolstad | Characterization of the chitinolytic machinery of Enterococcus faecalis V583 and high-resolution structure of its oxidative CBM33 enzyme[END_REF]. However, other strategies for AA10 LPMO production in the cytoplasm of E. coli with a cleavable N-terminal tag [START_REF] Forsberg | Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases[END_REF][START_REF] Gregory | Activity, stability and 3-D structure of the Cu(II) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens[END_REF][START_REF] Forsberg | Cleavage of cellulose by a CBM33 protein[END_REF] or as secreted protein using Gram-positive bacteria (Brevibacillus choshinensis SP3 or Bacillus subtilis) [START_REF] Nakagawa | A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting aand b-chitin[END_REF][START_REF] Yu | Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption[END_REF], cyanobacteria (Synechococcus elongatus UTEX 2973) [START_REF] Russo | Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium[END_REF], or yeast (Pichia pastoris) [START_REF] Rodrigues | Recombinant expression of Thermobifida fusca E7 LPMO in Pichia pastoris and Escherichia coli and their functional characterization[END_REF] as expression host have also been successfully developed. Using E. coli as recombinant expression system, we chose the N-terminal pelB signal peptide to direct PlAA10 production to the periplasm. Accordingly, the codon-optimized gene coding for PlAA10 was cloned directly in frame with the pelB signal peptide in the vector pET-22b(+) to ensure proper Nterminal processing. PlAA10 was overexpressed in the periplasm of E. coli BL21(DE3) after an overnight induction with IPTG at low temperature (16°C). The periplasmic protein fraction was released by cold osmotic shock and apo-PlAA10 was purified by affinity chromatography over chitin resin. Overall, the average yield of pure protein (as determined SDS-PAGE) was about 8 mg per liter of culture.

The intrinsic fluorescence of tryptophan residues was used to probe the Cu(II) binding to PlAA10. Upon titration of the apo-enzyme with Cu(II), tryptophan fluorescence of PlAA10 is linearly quenched (Figure S1a) as already observed for SliAA10E, a chitin-active LPMO from

Streptomyces lividans [START_REF] Chaplin | Heterogeneity in the histidine-brace copper coordination sphere in Auxiliary Activity Family 10 (AA10) lytic polysaccharide monooxygenases[END_REF]. A break point is reached at about 1.0-1.2 equivalent of Cu(II) after which the fluorescence remains constant (Figure S1b). These results are indicative of a 1:1 stoichiometry for the formation of the Cu(II)-PlAA10 complex, and also reveal a high affinity of the enzyme for its Cu(II) cofactor. Since the titration was carried out with 1 µM of apoenzyme, it implies a dissociation constant for Cu(II) at least ten times less than the protein concentration i.e. below 100 nM as expected for a bacterial LPMO. For instance, Kd values for Cu(II) in the low-to mid-nanomolar range were determined for only three chitin-active AA10

LPMOs: 55 ± 8 nM, 62.5 ± 0.2 nM, and from 6 to 80 nM (from pH 5 to 7) for SmAA10, SliAA10E, and BaAA10, respectively [START_REF] Gregory | Activity, stability and 3-D structure of the Cu(II) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens[END_REF][START_REF] Aachmann | NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions[END_REF][START_REF] Hemsworth | The copper active site of CBM33 polysaccharide oxygenases[END_REF][START_REF] Chaplin | Heterogeneity in the histidine-brace copper coordination sphere in Auxiliary Activity Family 10 (AA10) lytic polysaccharide monooxygenases[END_REF]. Although we did not measure the exact dissociation constant for Cu(II) in the case of PlAA10, the presence of a bulkier isoleucine in the second coordination sphere does not drastically impair copper binding. When needed, the holoenzyme was prepared by incubation of the apoenzyme with Cu(II) (5 equivalents) and thorough removal of the excess by two consecutive buffer exchanges.

The environment of the Cu(II) ion in PlAA10 was further studied by continuous wave EPR.

The X-band EPR spectrum of Cu(II)-PlAA10 recorded at 120K in the presence of 10% (v/v) glycerol (Figure 3) is similar in shape to that of other chitin-active LPMOs [START_REF] Forsberg | Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases[END_REF][START_REF] Gregory | Activity, stability and 3-D structure of the Cu(II) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens[END_REF][START_REF] Hemsworth | The copper active site of CBM33 polysaccharide oxygenases[END_REF][START_REF] Chaplin | Heterogeneity in the histidine-brace copper coordination sphere in Auxiliary Activity Family 10 (AA10) lytic polysaccharide monooxygenases[END_REF]. In particular, the rhombicity of the spectrum (gz ≠ gy ≠ gx) is indicative of a distorted geometry intermediate between square pyramidal and trigonal bipyramidal coordination geometries. In the parallel region, the spin Hamiltonian parameters were determined: gz = 2.260 and |Az| = 355

MHz (112 G). PlAA10 clusters with other chitin-active AA10 LPMOs and more closely to SmAA10 using the determined gz and |Az| parameters on a Peisach-Blumberg plot [START_REF] Peisach | Structural implications derived from the analysis of electron paramagnetic resonance spectra of natural and artificial copper proteins[END_REF][START_REF] Hemsworth | Production and spectroscopic characterization of lytic polysaccharide monooxygenases[END_REF]. As previously noted, the EPR signal of LPMOs can be affected by the presence or absence of glycerol as glassing agent [START_REF] Sabbadin | An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion[END_REF][START_REF] Hemsworth | Production and spectroscopic characterization of lytic polysaccharide monooxygenases[END_REF]. Accordingly, the EPR signal of PlAA10 is more complex in the absence of glycerol and clearly shows the contribution of two species (Figure S2). This complex EPR spectrum could be fitted with a major species (~ 80%) corresponding to that observed in the presence of glycerol (as in Figure 3) and a minor species (~ 20%). The latter exhibits a slightly lower gz value (2.235) but a significantly higher |Az| parameter (547 MHz, 175G) than the major species. However, further spectroscopic analysis such as Q-band EPR would be required to unambiguously assign the full spin Hamiltonian parameters of the Cu(II) ion for both species. The presence of several EPR signals was already reported for another chitin-active AA10 LPMO, SliAA10E. The second species also exhibits a significantly higher |Az| parameter and was proposed to originate from the discoordination of the N-terminal amine [START_REF] Chaplin | Heterogeneity in the histidine-brace copper coordination sphere in Auxiliary Activity Family 10 (AA10) lytic polysaccharide monooxygenases[END_REF]. Heterogeneity in the histidine brace by ligand discoordination could occur during freezing of the sample in the absence of glassing agent [START_REF] Hemsworth | Production and spectroscopic characterization of lytic polysaccharide monooxygenases[END_REF].

PlAA10 is a C1-oxidizing LPMO active on both α-and β-chitin

PlAA10 is a putative chitin-active LPMO according to multiple sequence alignment with other characterized AA10 LPMOs and corresponding phylogenetic analysis (Figure 2).

Additionally, chitin-binding by PlAA10 was verified as it was purified by affinity chromatography over chitin resin. Therefore, we evaluated PlAA10 activity towards both αand β-chitin. These two chitin allomorphs differ in the relative arrangement of polymeric chains: anti-parallel for α-chitin and parallel for β -chitin. PlAA10 was incubated with either α-or β-chitin in the presence of ascorbic acid as reductant under aerobic conditions i.e. with dioxygen as co-substrate. PlAA10 exhibits activity towards both α-and β-chitin. Accordingly, MALDI-TOF MS analysis of the reaction supernatant led to the detection of soluble oxidized chitooligosaccharides from degree of polymerization (DP) 2 to 6 and from 3 to 10 for α-and βchitin, respectively (Figure 4a). A similar product distribution is obtained whether the reaction was run in an unbuffered solution (Figure 4a) or at different pH values (5.0, 6.5, and 8.0, Figure S3 and S4). For each DP, three peaks are observed in the MS spectrum (Figure 4b). The first isotopic pattern corresponds to the aldonolactone C1-oxidization product (as Na + adduct), and the second and third to the aldonic acid (as Na + adduct or deprotonated with two Na + , respectively) resulting from the hydrolysis of the aldonolactone (Figure 4c). Further MS/MS confirmed the identification of the aldonolactone and its related aldonic acid (Figure S5), excluding any product resulting from C4 oxidation. Therefore, PlAA10 is a C1-oxidizing LPMO active on both α-and β-chitin. The C1-oxidation regiospecificity is a hallmark of chitinactive LPMOs [START_REF] Vaaje-Kolstad | An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[END_REF][START_REF] Hemsworth | Discovery and characterization of a new family of lytic polysaccharide monooxygenases[END_REF][START_REF] Sabbadin | An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion[END_REF][START_REF] Forsberg | Comparative study of two chitin-active and two cellulose-active AA10-type lytic polysaccharide monooxygenases[END_REF][START_REF] Nakagawa | A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting aand b-chitin[END_REF][START_REF] Gregory | Activity, stability and 3-D structure of the Cu(II) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens[END_REF]. According to MS data, oxidized products with even DP are preferred over odd DP for each substrate (Figure 4a) [START_REF] Vaaje-Kolstad | An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides[END_REF]. This preference derives from the structure of chitin with a two-fold screw axis along the polymer chain, leaving one every two glycosidic bonds of a surface polysaccharide chain buried inside the crystalline chitin and consequently inaccessible to the enzyme.

PlAA10 adopts the canonical immunoglobulin-like β-sandwich fold

To gain further insights into the influence of I113, we determined the X-ray crystal structure of PlAA10 at 1.6 Å resolution. PlAA10 crystallized in space group P212121 with three protein molecules in the asymmetric unit (Table 1). The crystal structure of PlAA10 reveals a threestranded and a four-stranded β-sheet forming a β-sandwich and displaying an overall immunoglobulin-like fold typical for LPMOs. Notably, a protrusion domain consisting of mainly loops, small helices, and two short β-strands is appended to the core β-sandwich (Figure 5a). This adjacent region exhibits the most variability among LPMOs [START_REF] Vaaje-Kolstad | Structural diversity of lytic polysaccharide monooxygenases[END_REF]. A first disulfide bridge (C39-C50) is found within this protrusion domain, while the second one (C146-C163) interconnects the two β-sheets of the core β-sandwich (Figure 5a). The overall structure of PlAA10 is similar to that of SmAA10 (PDB accession code 2BEM) with root-mean-square deviation (rmsd) values ranging from 0.73 to 0.76 Å for 169 aligned Cα atoms for monomers A, B, and C (monomer A as reference for SmAA10). In addition to the active site copper ion coordinated by H26 and H115 forming the so-called histidine brace motif (geometry described below), a second copper site is found between monomers A and B of two adjacent asymmetric units (Figure S6a). This copper-mediated crystal contact involves surface residues H83 (monomer A), D30 (monomer B), E130 (monomer B), and an alternative conformation of Q108 (monomer B) (Figure S6b). This second metal-binding site is probably due to the presence of excess Cu(II) ions in the protein buffer used for crystallization.

I113 does not affect the active site copper coordination geometry but protrudes from the substrate-binding surface

Full occupancy copper ions are found within the active site of PlAA10. The histidine brace coordination geometry around the copper is consistent with EPR data and similar to that of other chitin-active bacterial AA10 LPMOs (Table S2) [START_REF] Ciano | Bracing copper for the catalytic oxidation of C-H bonds[END_REF]. Namely, the Cu-NH2(H26) distance (2.2-2.3 Å) is indicative of a mixed Cu(II)/Cu(I) redox state. This is further supported by the presence of partially occupied (~ 40%) water molecules coordinating the active site copper ion (monomer A). Partial reduction of the native active Cu(II) ion of PlAA10 occurred during Xray diffraction data collection. Indeed, X-ray-induced photoreduction of the five-coordinate Cu(II) exhibiting a distorted geometry intermediate between a square pyramid and a trigonal bipyramid leads to a reduced three-coordinate T-shaped Cu(I) center after discoordination of the two water molecules as previously reported [START_REF] Hemsworth | The copper active site of CBM33 polysaccharide oxygenases[END_REF][START_REF] Gudmundsson | Structural and electronic snapshots during the transition from a Cu(II) to Cu(I) metal center of lytic polysaccharide monooxygenase by X-ray photoreduction[END_REF]. The absorbed dose of 178 kGy lies in the beginning of the dose scale probed by Gundmundsson et al. [83 kGy -1.07 MGy] to visualize the complete reduction of the Cu(II) ion [START_REF] Gudmundsson | Structural and electronic snapshots during the transition from a Cu(II) to Cu(I) metal center of lytic polysaccharide monooxygenase by X-ray photoreduction[END_REF]. Interestingly, the copper first coordination sphere is different in monomers B and C with the coordination of an extra ligand from a monomer of an adjacent asymmetric unit. Due to crystal contacts, the C-terminal carboxyl group of A199 (in monomers A and B) binds to the active site copper ion (in monomers C and B, respectively) (Figure S7), as observed in the X-ray crystal structure of the bacterial chitin-and cellulose-active AA10 LPMO (MaAA10B) from Micromonospora auriantica [START_REF] Forsberg | Structural determinants of bacterial lytic polysaccharide monooxygenase functionality[END_REF]. A similar coordination mode is also noticed in the structure of the fungal chitinactive AA11 LPMO from Aspergillus oryzae or that of the viral AA10 fusolin dimer from Melolontha melolontha entomopoxvirus, in which the active site copper ion is coordinated by the side chain of a neighboring glutamate residue in the crystal lattice or in the fusolin dimer, respectively [START_REF] Hemsworth | Discovery and characterization of a new family of lytic polysaccharide monooxygenases[END_REF][START_REF] Chiu | Structural basis for the enhancement of virulence by viral spindles and their in vivo crystallization[END_REF].

The side chain of I113 adopts a single conformation as shown by a clear electron density in each monomer (Figure 5b). Carbon Cβ of I113 is positioned almost identically to carbon Cβ of the conserved alanine in some chitin-active AA10 LPMOs (Figure 5c), pointing towards the copper and the exterior of the substrate-binding surface. Specifically, carbon Cβ of I113 is 3.9 Å away from the copper ion similarly to that of the alanine residue in other chitin-active AA10 LPMOs (3.8-4.2 Å). A superimposition of all the structures from bacterial chitin-active AA10 LPMOs determined to date shows that there is some slight variation regarding the relative position of the alanine side chain between enzymes (1.0 and 1.1 Å between the corresponding Cα carbons and Cβ carbons, respectively, of the two most dispersed alanine residues between aligned structures, Figure S8a). More variability is observed for bacterial cellulose-active AA10 LPMOs, although fewer X-ray crystal structures have been determined so far. Accordingly, the loop containing the conserved alanine can adopt significantly different conformations between enzymes [START_REF] Forsberg | Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases[END_REF][START_REF] Forsberg | Structural determinants of bacterial lytic polysaccharide monooxygenase functionality[END_REF]. Consequently, the position of side chain methyl group for this residue can be quite different (2.4 and 2.1 Å between the corresponding Cα carbons and Cβ carbons, respectively, of the two most dispersed alanine residues between aligned structures, Figure S8b) even though its distance to the active site copper ion stays within 3.5-4.0 Å. It was further shown that the alanine residue in bacterial cellulose-active LPMOs is not a determinant of oxidation regioselectivity [START_REF] Forsberg | Structural determinants of bacterial lytic polysaccharide monooxygenase functionality[END_REF].

The topology of the surface surrounding the active site is essential as it allows substrate binding. For chitin-active LPMOs, interactions with the substrate are mostly mediated by polar residues and a single aromatic amino acid (tyrosine, and sometimes tryptophan) from the substrate-binding surface [START_REF] Aachmann | NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions[END_REF][START_REF] Vaaje-Kolstad | Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21[END_REF][START_REF] Vaaje-Kolstad | Characterization of the chitinolytic machinery of Enterococcus faecalis V583 and high-resolution structure of its oxidative CBM33 enzyme[END_REF][START_REF] Chaplin | Heterogeneity in the histidine-brace copper coordination sphere in Auxiliary Activity Family 10 (AA10) lytic polysaccharide monooxygenases[END_REF]. Most amino acids important for chitin-binding by SmAA10, as determined by site-directed mutagenesis and NMR, are conserved in PlAA10 (PlAA10 residue numbering: Y55, E56, S59, E61, H115, T117, D183, and N186) [START_REF] Aachmann | NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions[END_REF][START_REF] Vaaje-Kolstad | Crystal structure and binding properties of the Serratia marcescens chitin-binding protein CBP21[END_REF].

However, some residues from the surface that were identified in SmAA10 to interact with chitin differ in PlAA10: K54, I111, M112, and I113, instead of Q53, L110, T111, and A112 in SmAA10 [START_REF] Aachmann | NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions[END_REF]. T111 and A112 in SmAA10 are in close contact with chitin as probed by molecular dynamics [START_REF] Bissaro | How a lytic polysaccharide monooxygenase binds crystalline chitin[END_REF]. Notably, the side chain hydroxyl group of T111 in SmAA10 hydrogen bonds with the acetyl group of an adjacent N-acetylglucosamine unit of chitin. This interaction will be abolished in PlAA10 with the methionine substitution. However, given the side chain orientation of M112 in the crystal structure of PlAA10 relative to T111 in SmAA10, it might not affect substrate binding. On the contrary, the side chain of A112 in SmAA10 is directly oriented towards chitin and its substitution by a bulkier isoleucine in PlAA10 is likely to cause a steric clash. For instance, the side chain of I113 protrudes from the chitin-binding surface in the crystal structure (Figure 5d) while it is relatively flat in other bacterial chitin-active AA10 LPMOs (Figure S9). The side chain of I113 will most likely adopt a conformation different from that observed in the crystal structure upon substrate binding but still remains bulkier than an alanine. Accordingly, the chitin-PlAA10 interaction is expected to be locally affected close to the active site while it is globally preserved thanks to all other conserved residues important for substrate binding distributed over the chitin-binding surface.

Bacterial AA10 LPMOs exhibit some variability at the active-site alanine position

To date, all the previously reported characterized chitin-active bacterial AA10 LPMOs display the conserved alanine residue in the second coordination sphere of the active site Cu(II) ion. The cellulose-active LPMOs CjAA10B from Cellvibrio japonicus, HcAA10A from Hahella chejuensis, and TtAA10 from Teredinibacter turnerae (all belonging to subclade IV, Figure 2a) are the only characterized bacterial AA10 LPMOs without the active site alanine, but substituted by a glycine instead [START_REF] Gardner | Systems biology defines the biological significance of a redox-active proteins during cellulose degradation in an aerobic bacterium[END_REF][START_REF] Ghatge | Multifunctional cellulolytic auxiliary activity protein HcAA10-2 from Hahella chejuensis enhances enzymatic hydrolysis of crystalline cellulose[END_REF][START_REF] Fowler | Discovery, activity and characterization of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae[END_REF]. Additionally, a crystal structure of a putative AA10 LPMO from Burkholderia pseudomallei with a methionine in place of the alanine was released in the Protein Data Bank (PDB accession code 3UAM). Nevertheless, no functional data are available and sequence analysis indicates that the first 18 N-terminal residues (i.e. before the conserved N-terminal histidine) are not predicted as a signal-peptide by SignalP 5.0 [START_REF] Armenteros | SignalP 5.0 improves signal peptide predictions using deep neural networks[END_REF] and Phobius [START_REF] Käll | Advantages of combined transmembrane topology and signal peptide prediction -the Phobius web server[END_REF]. Accordingly, the full protein was crystallized and no metal-coordinating histidine brace motif is observed in the crystal structure. PlAA10 is the first functional bacterial AA10 LPMO with a bulkier amino acid in place of the alanine to be biochemically and structurally characterized.

We explored potential substitutions at the conserved active-site alanine position among bacterial AA10 LPMOs annotated in the CAZy database. Multiple sequence alignment revealed some variability at this position. As expected, most of the putative bacterial AA10 LPMOs (~ 85%, 3762 out of the 4419 analyzed sequences) display the conserved alanine residue (Table S3). However, conservation at this position is not as high as previously thought. Different types of amino acids were found to replace the second coordination sphere alanine in putative bacterial AA10 LPMO including the less bulky glycine residue as in CjAA10B, HcAA10A, and TtAA10 (24 sequences); hydrophobic residues such as isoleucine (78 sequences) and valine (59 sequences); polar residues such as threonine (278 sequences), glutamine (58 sequences), lysine (10 sequences), serine (2 sequence), and arginine (1 sequence); and metal-coordinating residues such as methionine (143 sequences) and glutamate (4 sequences).

Interestingly, most of the putative isoleucine-containing AA10 LPMOs belong to the membrane-associated subclade (Figure 2a, subclade VI), the activity of which remains to be established although one member was shown to bind crystalline cellulose [START_REF] Walter | Characteristics of the surface-located carbohydratebinding protein CbpC from Streptomyces coelicolor A3(2)[END_REF]. Only few members are found within the chitin-active subgroup (Figure 2a, subclade I). Notably, these putative chitin-active AA10 LPMOs share 89-100% sequence identity with PlAA10 (mature protein) and all come from the genus of Gram-negative bacteria Photorhabdus: the DSPV002N strain of Photorhabdus luminescens, different strains of Photorhabdus luminescens subsp. laumondii (TT01 and an associated rifampicin-resistant laboratory strain, and DJC), Photorhabdus asymbiotica subsp. asymbiotica, and Photorhdabdus thracensis. Overall, this analysis unveils additional unusual second coordination sphere residues in place of the conserved alanine in other putative bacterial AA10 LPMOs.

Conclusions

Herein we biochemically and structurally characterized the single LPMO -PlAA10 -from the insect pathogenic bacterium Photorhabdus luminescens. PlAA10 was predicted to be active on chitin based on a phylogenetic analysis but sequence alignment revealed it lacks the conserved alanine residue in the close vicinity of the active site copper ion and on the substrate binding surface. Instead, the alanine is substituted by a bulkier isoleucine. We evidenced that PlAA10 binds Cu(II) similarly to other bacterial chitin-active LPMOs and that it is catalytically active on chitin (α-and β-forms) with a C1-oxidation regiospecificity. Its 1.6 Å resolution Xray crystal structure confirmed that PlAA10 adopts the immunoglobulin-like β-sandwich fold typical for LPMOs. Its overall structure is similar to that of other chitin-active AA10 LPMOs and includes a patch of conserved residues on the surface surrounding its active site that are essential for chitin binding. The copper coordination geometry is not altered by the nearbyisoleucine side chain, although it potentially remodels the substrate-binding surface close to the active site.

In closing, PlAA10 is the first AA10 LPMOs with a bulkier substitution of the conserved active site alanine residue to be characterized. It is interesting to consider the relative variability at the conserved alanine position in the active site of AA10 LPMOs as revealed by a bioinformatic analysis. Substitution by hydrophobic, polar, or even potentially metal coordinating residues provides some diversity close to the copper active site and on the substrate-binding surface. It is expected that these substitutions will alter in some extent enzyme activity and / or enzyme binding to its solid substrate. Future studies will address these effects on AA10 LPMOs catalytic activity and substrate binding and describe the characterization of other bacterial LPMOs with unusual second coordination sphere.

Materials and Methods

Protein sequence analysis

The signal peptide of the protein (GenBank accession number CAE14645.1, UNIPROT accession number Q7N4I5) encoded by plu2352 was determined and confirmed with webservers SignalP 5.0 [START_REF] Armenteros | SignalP 5.0 improves signal peptide predictions using deep neural networks[END_REF] and Phobius [START_REF] Käll | Advantages of combined transmembrane topology and signal peptide prediction -the Phobius web server[END_REF]. Multiple sequence alignment of PlAA10 (residues 26 to 199) along with the catalytic domain of 32 other characterized and putative bacterial AA10 LPMOs (Table S1) was performed with Clustal Omega [START_REF] Sievers | Fast scalable generation of highquality protein multiple sequence alignments using Clustal Omega[END_REF]. An unrooted phylogenetic tree was built from this alignment and visualized using the web-based tool Interactive Tree Of Life [START_REF] Letunic | Interactive Tree Of Life (iTOL): an online tool for phylogenetic tree display and annotation[END_REF].

GenBank accession numbers for all bacterial AA10 LPMOs referenced in the CAZy database (4549 as of 2019, September 1 st ) were used to retrieve the corresponding amino acid sequences in NCBI. A total of 4532 sequences (16 were found as duplicates and one removed from GenBank) were subsequently aligned with Clustal Omega [START_REF] Sievers | Fast scalable generation of highquality protein multiple sequence alignments using Clustal Omega[END_REF]. 113 sequences lacking either one of the copper(II)-coordinating histidine residues or the conserved axial phenylalanine / tyrosine residue were excluded from the analysis. The remaining 4419 sequences were classified according to the nature of the amino acid at the conserved active site alanine position (Table S3).

Bacterial DNA sequence analysis

Tryptic soy broth (TSB, 5 mL) was inoculated with a Photorhabdus luminescens subsp. (GE Healthcare) with 50 mM MES (pH 6.5) as storage buffer. Apo-PlAA10 was prepared similarly i.e. two successive buffer exchanges in the same storage buffer after the purification by affinity chromatography. After concentration, apo-and holo-PlAA10 were stored at -80°C.

Protein concentration was determined from the absorbance at 280 nm using a calculated extinction coefficient of 38180 M -1 cm -1 [START_REF] Gill | Calculation of protein extinction coefficients from amino acid sequence data[END_REF].

Fluorescence

Fluorescence spectroscopy was performed on a Horiba Jobin-Yvon Fluoromax-4 fluorimeter. Emission spectra following an excitation at 295 nm (1 nm excitation slit widths)

were recorded at 25°C between 300 and 450 nm (20 nm emission slit widths). Protein solution was prepared by dilution of a stock solution of apo-PlAA10 into 50 mM MES (pH 6.5), with a final protein concentration of 1 µM. Fluorescence was recorded upon protein titration with a solution of Cu(II)(NO3)2 (successive addition of 0.2 equivalent) after an equilibration of 2 min at 25°C.

Electron paramagnetic resonance (EPR) spectroscopy and simulation

Protein samples (holo-PlAA10 concentrations ranging from 150 to 200 µM) for continuous wave EPR were prepared in 50 mM MES buffer (pH 6.5) supplemented or not with 10% (v/v) glycerol. Buffers with and without glycerol were prepared independently and both adjusted to pH 6.5. EPR spectra were recorded on a Bruker Elexsys E500 spectrometer operating at Xband at 120 K (BVT 3000 digital temperature controller) with the following acquisition parameters: modulation frequency, 100 kHz; modulation amplitude, 5 G; conversion time, 120 ms; sweep time, 123 s; and microwave power, 20 mW. EPR spectra were simulated using the EasySpin toolbox developed for Matlab [START_REF] Stoll | EasySpin, a comprehensive software package for spectral simulation and analysis in EPR[END_REF].

Chitin cleavage assay

The activity of PlAA10 was assessed on both α-chitin from shrimp shell (practical grade, powder, Sigma Aldrich) and β-chitin from squid-pen (particle size < 500 µm, kind gift from Dr. D. Gillet, Gillet Chitosan SAS, France). 1 mL-reactions containing 5 µM PlAA10, 2.5 mg chitin, 1 mM ascorbic acid in deionized water (no buffer), or in 10 mM sodium acetate (pH 5.0), or in 10 mM MES (pH 6.5), or in 10 mM Tris (pH 8.0) were set up in capped 15 mL round-bottom polypropylene tubes. Samples were incubated horizontally at 37°C for 24 hours whilst rotating to ensure proper substrate mixing. Reaction supernatants were collected after centrifugation at 13000 rpm and filtrated over a 10 kDa MWCO centrifugal concentrator to remove any remaining enzyme before analysis of the filtrate by mass spectrometry (MS).

Mass spectrometry

Products resulting from enzyme reactions as described above were analyzed by MS. Two types of mass measurements were performed on the samples: firstly, a mass profile was recorded by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) MS.

Then, ions of interest were selectively analyzed by electrospray ionization (ESI) MS/MS upon a fragmentation by collision-induced dissociation (CID). For MALDI-TOF MS measurements, an ionic preparation of 2,5-dihydroxybenzoic acid (DHB) and N,N-dimethylaniline (DMA) was used as the MALDI matrix, according to Ropartz et al. [START_REF] Ropartz | Performance and evaluation on a wide set of matrix-assisted laser desorption ionization matrices for the detection of oligosaccharides in a high-throughput mass spectrometry screening of carbohydrate depolymerizing enzymes[END_REF]. Samples (1 µL) were deposited on a polished steel MALDI target plate and covered by the matrix (1 µL). After a complete drying of the deposits, MALDI measurements were performed on an Autoflex Speed MALDI-TOF/TOF spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser (355 nm, 1000 Hz) and controlled using the Flex Control 3.0 software package. The mass spectrometer was operated with positive polarity in a reflectron mode, and spectra were acquired in the range of 400-2500 m/z. For ESI MS/MS measurements, experiments were performed on a Synapt G2Si high-definition mass spectrometer (Waters Corp., Manchester, UK). Fragmentation was performed by CID in the transfer cell of the instrument, using appropriate collision energies depending on the precursor. Samples were diluted ten-fold in MeOH/H2O (1:1, v/v) and infused at a flow rate of 5 µL.min -1 . Acquisitions were conducted in positive polarity, as well as in 'sensitivity' mode.

Protein crystallization

Initial crystallization screening was performed with the two high-throughput sparse-matrix screening kits Index HT and Crystal (Hampton Research, USA) in sitting drops using the vapor diffusion method. In general, a 1 µL drop of precipitant was added to a 1 µL drop containing 5 or 10 mg/mL holo-PlAA10, 50 mM MES (pH 6.5), and 500 µM CuSO4 and equilibrated against a 80 µL reservoir of precipitant solution at 21°C. Among initial hits, needle and rod-like crystals were obtained with Crystal screen condition E11 consisting of 0.01 M cobalt chloride, 0.

Data collection and structure refinement

Prior to data collection, crystals were flash-cooled in liquid nitrogen after transfer to a cryoprotectant solution consisting of precipitant solution supplemented with 15% (v/v) glycerol. The best diffracting crystal (1.6 Å) was obtained as previously described with a 10 mg/mL protein solution (50 mM MES (pH 6.5), and 500 µM CuSO4) and a precipitant solution containing 0.1 M sodium acetate (pH 5.2) and 0.7 M 1,6-hexanediol. X-ray diffraction data were collected at the European Synchrotron Radiation Facility (ESRF, Grenoble, France) on beamline ID29 [START_REF] De Sanctis | ID29: a high-intensity highly automated ESRF beamline for macromolecular crystallography experiments exploiting anomalous scattering[END_REF] at 100 K at a wavelength of 0.976 Å. A continuous oscillation range of 101° was collected in 74.74 s with an X-ray flux of 4.48 10 10 photons.s -1 , resulting in an average diffraction-weighted dose absorbed by the exposed region of 178 kGy calculated using RADDOSE-3D available at http://raddo.se [START_REF] Zeldin | RADDOSE-3D: time-and space-resolved modelling of dose in macromolecular crystallography[END_REF]. Data were integrated and reduced using the autoPROC pipeline [START_REF] Vonrhein | Data processing and analysis with the autoPROC toolbox[END_REF] chosen from the series of automated reduction pipelines [START_REF] Monaco | Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF[END_REF], whose results are available through the web information management system ISPyB [START_REF] Delagenière | ISPyB: an information management system for synchrotron macromolecular crystallography[END_REF]. The structure belonged to space group P212121 with three protein molecules in the asymmetric unit.

The structure was solved by molecular replacement using PHASER [START_REF] Mccoy | Phaser crystallographic software[END_REF] implemented in PHENIX [START_REF] Adams | PHENIX: a comprehensive python-based system for macromolecular structure solution[END_REF] with the atomic coordinates (PDB accession code 2BEM) of the wild-type AA10

LPMO from Serratia marcescens (SmAA10, also known as CBP21) less ion, and solvent used as a search probe for rotation and translation function calculations. Each model was refined with iterative cycles of refinement in PHENIX [START_REF] Adams | PHENIX: a comprehensive python-based system for macromolecular structure solution[END_REF] and manual model rebuilding in COOT [START_REF] Emsley | Features and development of Coot[END_REF]. Ions and solvent molecules were added after several rounds of refinement. In the final stages, translation libration screw (TLS) refinement was performed. TLS groups were automatically determined using PHENIX [START_REF] Adams | PHENIX: a comprehensive python-based system for macromolecular structure solution[END_REF]. Anisotropic temperature factor refinement was performed at the last stage of refinement for all atoms except water molecules. Side chains of residues that were disordered were excluded from the final model and include E167 in monomer A; E167 in monomer B; and K96, E104, Q151, K153, and R158 in monomer C. Few electron density peaks that were not confidently interpretable were left unmodeled (such as close to D49 in monomer C). Detailed data collection and refinement statistics are recorded in Table 1.

Tables

Figure 1 .

 1 Figure 1. Reaction catalyzed by LPMOs and active site of bacterial AA10 chitin-active

Figure 2 .

 2 Figure 2. Sequence analysis of PlAA10 and other bacterial AA10 LPMOs. (a) Unrooted

Tf AA 10 A

 10 Photorhabdus luminescens subsp. laumondii (CAE14645.1) 105 IEFQW EIM-IQHKT--SSW Bacillus amyloliquefaciens (CBI42985.1) 115 NTFTW HYT-APHAT--SKW Bacillus licheniformis (AAU39477.1) 134 HTFTW TYT-APHNT--SQW Bacillus thuringiensis (AJP62637.1) 109 NTFKW KLT-APHST--KEW Cellvibrio japonicus (ACE83992.1) 127 E-FVW TNT-AAHKT--KYF Enterococcus faecalis (AAO80225.1) 104 LDITW NLT-AQHRT--ASW Jonesia denitrificans (ACV09037.1) 99 VDLTW KLT-APHNT--STW Listeria monocytogenes (CAD00545.1) 102 LTVEW TLT-APHKT--SSW Paenibacillus larvae (AFQ34195.1) 112 HTFTW TLT-APHRT--AKW Serratia marcescens (AAU88202.1) 104 NSFTW KLT-ARHST--TSW Streptomyces ambofaciens (CAJ89556.1) 96 ATFSW KLT-AQHST--STW Streptomyces griseus subsp. griseus (BAG23684.1) 96 QQFQW KLT-ARHAT--STW Streptomyces lividans (EOY47895.1) 110 YTFRW QFT-ARHAT--TDF Vibrio cholerae (AAF96709.1) 111 QTFEW TFT-ANHVT--KDW

Figure 3 .

 3 Figure 3. EPR spectroscopy of PlAA10. X-band EPR spectrum (plain line) of Cu(II)-PlAA10

Figure 4 .

 4 Figure 4. Activity of PlAA10 towards α-and β-chitin. (a) MALDI-TOF mass

Figure 5 .

 5 Figure 5. X-ray crystal structure of PlAA10. (a) Overall structure of PlAA10. The core beta

  laumondii TT01 (DSM 15139, DSMZ, Germany) glycerol stock (in TSB, stored at -80°C) and was cultured overnight at 28°C under agitation (200 rpm). After centrifugation at 5000 rpm at 4°C for 10 min, genomic DNA was extracted from the cell pellets using the DNeasy Blood and Tissue kit (QIAGEN) according to the manufacturer's protocol. A 1 kb fragment containing plu2352 was amplified from Photorhabdus luminescens genomic DNA by pcr using 5'-CTAGAATTGATAGTTAAGTCCTGGGGCTTTATTTGTCG-3' and 5'-agitation (200 rpm), and was used to inoculate 6 × 500 mL cultures (1% v/v inoculum in LB supplemented with 100 µg/mL ampicillin). Cells were grown at 37°C under agitation (200 rpm) until OD600 ~ 0.5 at which point the temperature was lowered to 16 °C. At OD600 ~ 0.7, cells were induced with isopropyl-β-D-thiogalactopyranoside (IPTG, 0.2 mM final concentration) and grown overnight (20 h) at 16 °C and 200 rpm. Cells were harvested by centrifugation at 4 °C (10 min at 5000 g). The periplasmic protein fraction was prepared by cold osmotic shock as previously described with slight modifications [53]. Briefly, the cell pellet was re-suspended in 100 ml (1/30 of the total culture volume) ice-cold sucrose buffer (50 mM Tris, 20% w/v sucrose, 1 mM EDTA, pH 8.0) and incubated at 4°C under gentle agitation (4 rpm on a tube rotator). After 1h, the cell suspension was supplemented with MgSO4 (60 µL of 1M MgSO4 solution for each gram of wet cell pellet) and incubated under the same conditions for 30 min. The supernatant was collected by centrifugation at 4°C (20 min at 20000 g) and stored at 4°C. The corresponding cell pellet was re-suspended in 100 mL (1/30 of the total culture volume) icecold milliQ water and further incubated under gentle agitation (4 rpm on a tube rotator) for 45 min at 4°C. The cell suspension was centrifuged (20 min at 20000 g, 4 °C) to recover the supernatant. The first supernatant fraction was dialyzed against 20 mM Tris (pH 8.0), 1 mM EDTA in 3.5 kDa molecular weight cut-off (MWCO) dialysis tubing (Spectra/Por, Spectrumlabs). The second supernatant fraction was supplemented with 20 mM Tris (pH 8.0). Both periplasmic fractions were adjusted to 1.0 M (NH4)2SO4 by slow addition of solid ammonium sulfate at 4°C, and filtered over a 0.22 µm membrane. Periplasmic extracts were loaded at 4°C onto a chitin bead column (NEB) pre-equilibrated with 20 mM Tris (pH 8.0), 1.0 M (NH4)2SO4 (running buffer). After washing with the running buffer, PlAA10 was eluted with 20 mM acetic acid. Fractions containing purified PlAA10 as determined by SDS-PAGE were adjusted with 70 mM Tris buffer (pH 8.0), pooled, and concentrated (10 kDa MWCO Vivaspin 20, Sartorius). Protein metalation was done at 4°C by the addition of a 5-fold molar excess of CuSO4. Excess Cu(II) was removed by two successive buffer exchanges over a PD-10 column

1 M

 1 sodium acetate trihydrate (pH 4.6), and 1.0 M 1,6-hexanediol. Crystallization was further optimized by a grid screen with the following precipitant conditions: 0 to 0.01 M cobalt chloride, 0.1 M sodium acetate (pH 4.3 to 5.2), and 0.70 to 1.15 M 1,6-hexanediol. Typically, crystals appeared after one to three days and grew to full dimensions (~ 100 x 200 x 700 µm) within a week.

Table 1 .

 1 Data collection and refinement statistics Values in parentheses refer to the highest shell of data. b Rmerge = åïIh -áIñhï/åIh, where áIñh is the average intensity for reflection h calculated from replicate reflections. c Pearson correlation coefficient random half-datasets. d Rcryst = åï|Fo| -|Fc|ï/å|Fo| for reflections contained in the working set. êFoê and êFcê are the observed and calculated structure factor amplitudes, respectively. e Rfree = åï|Fo| -|Fc|ï/å|Fo| for reflections contained in the test set held aside during refinement. f Per asymmetric unit. g Calculated with MolProbity.

	Wild-type PlAA10

a

Acknowledgements

CNRS and Aix Marseille Université are acknowledged for their financial support. AM is a recipient of a French Ministère de l'Enseignement Supérieur et de la Recherche PhD fellowship. The ESRF is acknowledged for access to beamline ID29 via its in-house research program.

primers, respectively. The pcr product was purified by electrophoresis on a 1% (w/v) agarose gel and extracted using the QIAquick Gel Extraction kit (QIAGEN) according to the manufacturer's protocol. The pcr product was partially sequenced (Genewiz, UK) using 5'-CTAGAATTGATAGTTAAGTCCTGGG-3' as sequencing primer and the plu2352 nucleotide sequence and corresponding amino acid sequence are reported in Supplementary Information Table S4.

Cloning

The synthetic codon-optimized DNA fragment (Life Technologies) coding for the catalytic domain of the putative LPMO from Photorhabdus luminescens subsp. laumondii TT01 (GenBank ID CAE14645.1, residues 26 to 199) was amplified by pcr using 5'-CTCCTCGCTGCCCAGCCGGCGATGGCCCATGGCTATATTGATAGTCCGGGTAG-3' and 5'-GTGGTGCTCGAGTGCGGCCGCAAGCTTTATGCCGGACTAATGGTGGTATCAATCA CC-3' as forward and reverse primers, respectively. The pcr product was purified by electrophoresis on a 1% (w/v) agarose gel and extracted using the QIAquick Gel Extraction kit (QIAGEN) according to the manufacturer's protocol. The insert was cloned in frame with the pelB signal peptide into the pET-22b(+) vector (Novagen) (double-digested with MscI and HindIII restriction enzymes (NEB)) using the sequence-and ligation-independent cloning method [START_REF] Jeong | One-step sequence-and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies[END_REF]. The corresponding pET-22b(+) SPpelB-PlAA10 plasmids from positive clones were sequenced (Genewiz, UK) to confirm the correct incorporation of the desired construct (nucleotide and amino acid sequences in Table S4).

Protein expression and purification

The

Conflicts of Interest

The authors declare no conflict of interest.

Supporting Information

Additional supporting information may be found online in the Supporting Information section at the end of the article.

Table S1. Origin, name, and GenBank accession number of all AA10 LPMOs sequences included in the unrooted phylogenetic tree.

Table S2. Geometry of the copper active site of PlAA10.

Table S3. Amino acid variability at the conserved alanine position in bacterial AA10 LPMOs referenced in the CAZy database and GenBank accession number of the corresponding sequences.

Table S4. Amino acid sequence of PlAA10 with its native signal peptide or the pelB signal peptide, and nucleotide sequence of the corresponding genes (plu2352 in Photorhabdus luminescens subsp. laumondii TT01 and the gene cloned in pET-22b(+)) Enterococcus faecalis with a conserved alanine residue and PlAA10.