Functional insights into a peculiar tetra-modular LPMO from the human pathogen Enterobacter cloacae

Functional insights into a peculiar tetra-modular LPMO from the human pathogen Enterobacter cloacae | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Functional insights into a peculiar tetra-modular LPMO from the human pathogen Enterobacter cloacae Koyel Bardhan , Saumashish Mukherjee , Theruvothu Madathil Vandhana , Alessia Munzone , Andres Posbeyikian , Sacha Grisel , Lal Duhsaki , Jean-Guy Berrin , View ORCID Profile Bastien Bissaro , View ORCID Profile Jogi Madhuprakash doi: https://doi.org/10.1101/2025.07.04.663164 Koyel Bardhan 1 Department of Plant Sciences, School of Life Sciences, University of Hyderabad , Gachibowli, Hyderabad-500046, Telangana, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Saumashish Mukherjee 1 Department of Plant Sciences, School of Life Sciences, University of Hyderabad , Gachibowli, Hyderabad-500046, Telangana, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Theruvothu Madathil Vandhana 1 Department of Plant Sciences, School of Life Sciences, University of Hyderabad , Gachibowli, Hyderabad-500046, Telangana, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alessia Munzone 2 INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques , 13009 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Andres Posbeyikian 3 The Sainsbury Laboratory, University of East Anglia , Norwich Research Park, NR4 7UH, Norwich, UK Find this author on Google Scholar Find this author on PubMed Search for this author on this site Sacha Grisel 2 INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques , 13009 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lal Duhsaki 1 Department of Plant Sciences, School of Life Sciences, University of Hyderabad , Gachibowli, Hyderabad-500046, Telangana, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jean-Guy Berrin 2 INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques , 13009 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site Bastien Bissaro 2 INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques , 13009 Marseille, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Bastien Bissaro For correspondence: jmpsl{at}uohyd.ac.in bastien.bissaro{at}inrae.fr Jogi Madhuprakash 1 Department of Plant Sciences, School of Life Sciences, University of Hyderabad , Gachibowli, Hyderabad-500046, Telangana, India Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jogi Madhuprakash For correspondence: jmpsl{at}uohyd.ac.in bastien.bissaro{at}inrae.fr Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Enterobacter cloacae is a Gram-negative nosocomial human pathogen that inhabits diverse ecological niches. Its genome encodes a conserved set of putative chitin-active enzymes, including a peculiar lytic polysaccharide monooxygenase (LPMO), termed Ec LPMO, which we functionally characterized in this study. Ec LPMO is a tetra-modular protein consisting of an auxiliary activity family 10 (AA10) catalytic domain, two central domains of unknown function (DUF-A and DUF-B), and a C-terminal carbohydrate-binding module (CBM73). Functional assays using full-length Ec LPMO and its truncated variants demonstrated that the AA10 domain oxidatively cleaves chitin at the C1 position. The CBM73 module enhances chitin binding and promotes synergy with endogenous chitinases. Notably, Ec LPMO displayed a particularly strong synergistic effect with the unimodular chitinase Ec ChiA, leading to up to 14-fold and 60-fold increases in GlcNAc release from α- and β-chitin, respectively. Deletion of both DUFs reduced Ec LPMO activity. While DUF-A alone and the association of DUF-A and DUF-B showed limited chitin binding, DUF-B alone exhibited no binding, suggesting a distinct role. Unexpectedly, using state-of-the-art structural modelling (AlphaFold3), we observed that the DUF-B domain contains two highly conserved histidines that coordinate the AA10-bound copper, forming a previously unreported ‘inter-domain tetra-histidine copper coordination’ center. These findings highlight the structural and functional complexity of Ec LPMO and suggest that its accessory domains, particularly DUF-B, may contribute to enzyme stability and substrate interaction. We speculate that DUF-B may protect the LPMO active site from oxidative damage, a feature that could prove crucial in its ecological and pathogenic contexts. 1. Introduction Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that mediate oxidative cleavage of glycosidic bonds in crystalline polysaccharides such as chitin ( 1 ) and cellulose ( 2 - 4 ). This oxidative mechanism introduces nicks on the polysaccharide surface, disrupting its structure and facilitating more efficient hydrolysis by glycoside hydrolases (GHs) ( 5 , 6 ). At their active site, LPMOs contain a mononuclear copper ion coordinated by two conserved histidine residues, known as the ‘histidine brace’ ( 7 ). To become catalytically active, the copper must first be reduced from Cu(II) to Cu(I), typically by external electron donors such as small organic compounds (e.g., ascorbate) or enzymatic partners ( 3 , 8 - 10 ). Once reduced, and in the presence of hydrogen peroxide, the LPMO-Cu(I) can then catalyze hydroxylation of either the C1 or C4 carbon of the scissile glycosidic bond, triggering cleavage via a peroxygenase-type mechanism ( 11 - 15 ). The resulting products are typically aldonic acids (C1-oxidation) or geminal diols (C4-oxidation). LPMOs are currently grouped in the Carbohydrate-Active enZymes (CAZy) database as Auxiliary Activities (AA), spanning eight families (AA9–11 and AA13–17) ( 16 ). Chitin-active LPMOs are primarily found in families AA10 ( 1 ), AA11 ( 17 ), and AA15 ( 18 ). While AA11s are restricted to fungi, AA15 LPMOs occur across viruses, algae, oomycetes, and insects. The AA10 family stands out as the most taxonomically diverse, with representatives from bacteria ( 1 ), viruses ( 19 , 20 ), plants ( 21 ), and, as more recently unveiled, fungi ( 22 ). Of note, most of these AA10 LPMOs characterized to date act on chitin. Similar to other CAZymes, many LPMOs are multi-modular, incorporating additional domains such as carbohydrate-binding modules (CBMs), GH domains, or glycosylphosphatidylinositol (GPI) anchors. These domains can modulate LPMO binding affinity, catalytic efficiency, and stability ( 23 ). They are typically connected by flexible linkers that influence interdomain interactions and substrate accessibility ( 24 ). In addition, many LPMOs contain disordered C-terminal regions (dCTR), which are present across most LPMO families (except AA13), and still remain largely functionally uncharacterized ( 25 ). Regarding AA10 LPMOs, to date, approximately 23 multi-modular LPMOs have been experimentally characterized for activity on chitin or cellulose. Beyond their recognized role in biomass degradation, LPMOs are now being implicated in diverse biological functions ( 26 ), including pathogenesis and cellular development ( 27 - 31 ). Notably, several bacterial AA10 LPMOs have been linked to virulence. One early example is GbpA from Vibrio cholerae , a chitin-active LPMO involved in mucin binding and intestinal colonization ( 32 ). More recently, CbpD from Pseudomonas aeruginosa was identified as a key virulence factor supporting bacterial survival in the human bloodstream ( 27 ). In the course of identifying LPMO candidates from human-associated bacterial pathogens, we focused on the Gram-negative nosocomial pathogen Enterobacter cloacae. E. cloacae is a clinically relevant opportunistic pathogen associated with hospital-acquired infections, particularly in immunocompromised individuals. It can cause respiratory, urinary tract infections, and meningitis ( 33 ). Due to its increasing antibiotic resistance, it is classified among the WHO-designated ESKAPE pathogens ( 34 ). Genome analysis revealed a conserved, single-copy, multi-modular AA10 LPMO across 46 complete E. cloacae genomes (NCBI; Table S1 ), which we refer to as Ec LPMO. Its modular organization shows resemblance to previously studied virulence-associated LPMOs such as GbpA (from V. cholerae ; 32), CbpD (from P. aeruginosa; 27) and Vca LPMO10A (from V. campbellii ; 35), despite relatively low sequence identity. This conservation and structural similarity suggest that Ec LPMO may play a biologically important role, potentially linked to the host-pathogen interface. In this study, we aimed to characterize the chitinolytic machinery of E. cloacae , with a specific focus on Ec LPMO. Using sequence and structural in silico analyses, we predicted functional properties and designed truncated protein variants. We then assessed the ability of Ec LPMO and its variants to bind and oxidize crystalline chitin, providing insight into the role of its accessory domains. Finally, we examined the synergistic effects of Ec LPMO with E. cloacae chitinases on α-and β-chitin, advancing our understanding of how this enzyme may contribute to efficient chitin degradation and potentially to pathogenicity. 2. Materials and methods 2.1. Bioinformatic analysis The annotation of chitin-active CAZymes for different strains of E. cloacae (including the one under study, i.e., ATCC 13047 T ) was conducted using the dbCAN3 meta-server ( 36 ). An integrated approach using all three designated tools/databases of the dbCAN3 server i.e., HMMER:dbCAN, Diamond:CAZy and HMMER:dbCAN-sub, was employed for CAZyme prediction and annotation. Only those proteins or domains annotated by at least two tools were considered for further analysis ( 36 ). We also present the frequency of strains that possess ‘n’ members of a given CAZy family, illustrating the distribution of chitin-active CAZymes. ( Table S2 ). Domain architecture of Ec LPMO and few other chitin-active CAZymes ( Ec ChiA, Ec ChiB, Ec ChiC and Ec HexA) from E. cloacae ATCC 13047 T was predicted using Pfam ( https://pfam.xfam.org/ ) and the NCBI Conserved Domain Database https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml ). SignalP 5.0 ( http://www.cbs.dtu.dk/services/SignalP/ ) was used to predict the presence of signal peptide. Genetic arrangement and genomic localization of these CAZymes in the representative type strain genome, E. cloacae ATCC 13047 T was identified using the RAST servers. The modularity of Ec LPMO was further analyzed by the expert annotation of Prof. B. Henrissat (CAZy database, Marseille, France), who determined the boundaries of the different domains (i.e. AA10, DUF-A, DUF-B and CBM73). Sequence alignment of Ec LPMO with its homologs was performed using the T-Coffee server ( https://tcoffee.crg.eu/ ) and the output was generated using ESPript 3.0. 2.2. Phylogenetic analysis To build the phylogenetic tree of AA10s, we used 176 AA10 sequences including sequences belonging to clades with characterized members, and sequences belonging to clades still uncharacterized. Of note, the N-terminal signal peptide and the variable C-terminal regions of all AA10 sequences were cut manually using BioEdit ( 37 ) in order to keep the catalytic domain only. The non-redundancy of sequences was verified using CD-HIT. The 176 AA10 sequences were aligned using MAFFT-DASH (L-INS-i method) ( 38 ), which includes structural data input. The resulting multiple sequence alignments were used to infer the phylogenetic trees via the IQ-Tree webserver ( 39 ), with the standard bootstrap analysis (100 iterations) and a SH-aLRT branch test (1,000 replicates). To build the phylogenetic tree of CBM73s, 5,025 sequence IDs of CBM73-containing proteins were retrieved from the CAZy database, which were used to retrieve the sequences from the NCBI database. The sequences were aligned with MAFFT-FFT-large-NS-2 ( 38 ) and, upon inspection of (predicted/experimental) structures, the region of about 60 amino acids corresponding to the CBM73 ( 40 ) was extracted from the MSA using BioEdit software. To decrease the number of sequences in order to build a reliable phylogenetic tree, CBM73 proteins sharing a sequence identity > 90% were clustered using CD-HIT ( 41 ), resulting in 572 clusters. A representative sequence of each cluster (provided by the CD-HIT software) was used to build the phylogenetic tree. This pool of sequences includes sequences of characterized CBM73, i.e. , the CBM73s from Pseudomonas aeruginosa PA01 (GenBank ID AAG04241.1, UniProt ID Q91589) ( 42 ), from Cellvibrio japonicus Ueda107 (GenBank ID ACE83992.1, UniProt ID B3PJ79, (NMR) PDB ID 6Z41) ( 43 ) and from Vibrio campbellii ATCC BAA-1116 (GenBank ID ABU72648.1, PDB ID 8GUM) ( 35 ). To build the tree, the 572 sequences were aligned with MAFFT v7 and the resulting multiple sequence alignment was used to infer the phylogenetic trees via the IQ-Tree webserver ( 39 ), with the ultrafast bootstrap analysis (1,000 iterations) and a SH-aLRT branch test (1,000 replicates). Each branch was then assigned with SH-aLRT and UFBoot supports, allowing to reliably define clades when SH-aLRT ≥ 80% and UFBoot ≥ 95%. Sequences forming each clade were retrieved and re-aligned with MAFFT v7 to generate a sequence conservation plot using WebLogo 3.0 ( 44 ). The phylogenetic trees were visualized in iTOL ( 45 ) and edited in Illustrator CC 2017. 2.3. Structure determination using AlphaFold Mature wild-type Ec LPMO and Vc GbpA were modelled using AlphaFold3 via the AlphaFold Server ( 46 ). Corresponding histidine-to-alanine variants ( Ec LPMO H354A,H384A and Vc GbpA H357A,H388A ) were also modelled. For both wild-type and mutant proteins, structural predictions were generated either in the apo form or in complex with Cu(II). Default template mode was used, with the PDB template cutoff set to 30/09/2021, and model generation was repeated across five fixed seed values (Seed = 1–5). A custom bash script was used to extract confidence metrics from the AlphaFold server output, including pTM, ipTM, and PAE plots, and to generate box plots of ipTM scores across the five seed replicates. Structural models were visualized using UCSF ChimeraX ( 47 ). A custom Python script was written to identify protein atoms located within 3.6 Å of the modelled copper ion, extracting all atomic contacts for each predicted structure. This data was used to generate UpSet plots in Python, summarizing how many seed replicates (out of 5) predicted contacts between Cu(II) and the four conserved histidine residues: His22, His109, His354, and His384 for Ec LPMO, and His24, His121, His357, and His388 for Vc GbpA. 2.4. Cloning, expression and purification of recombinant enzymes The full-length LPMO gene, Ec LPMO FL (residues 1-484) including the native signal peptide (residues 1-21) was amplified from the genomic DNA of E. cloacae ATCC 13047 using the primers listed in Table S3. Both the insert and pET-22b(+) expression vector were double digested using the restriction endonucleases Nde I and Xho I (Thermo Scientific, USA), and were ligated using the Rapid Ligation kit (ThermoScientific, USA). The entire ligation mixture was then transformed by heat-shock into chemically competent E. coli DH5α cells. Positive clones were screened using colony PCR and the sequence integrity was further confirmed by Sanger sequencing. The same strategy was followed for generating the truncations of Ec LPMO FL , which were, Ec LPMO ΔCBM73 (residues 1-413), Ec LPMO CD (residues 1-190), the DUFA-DUFB double domains (residues 205-413), and the single domain DUF-A (residues 205-308), DUF-B (residues 316-413) and CBM73 (residues 424-484) using the primer combinations as listed in the Table S3. Sequence-validated clones were transformed by heat-shock into chemically competent BL21 (DE3) pLysS E. coli cells. Colonies obtained were inoculated into lysogenic broth (LB) containing 100 μg/mL ampicillin. Ec LPMO FL , Ec LPMO ΔCBM73 , DUF-A and CBM73 were expressed by growing the transformants at 37°C for 3 h (until the OD reached 0.4-0.6) followed by induction with 0.4 mM IPTG for 16 h at 18°C and shaking at 180 rpm. A similar strategy was followed for the double domain DUFA-DUFB and the single domain DUF-B, but induction was performed with 0.7 mM IPTG. Ec LPMO CD was expressed by growing at 30°C for 16 h without IPTG induction. The cells were harvested by centrifugation at 8000 rpm for 15 min. All the AA10-containing variants, i.e. Ec LPMO FL , Ec LPMO ΔCBM73 and Ec LPMO CD , all contained the native signal peptide and were thus isolated from the periplasmic fraction (SP cleaved off upon export) using an osmotic shock method essentially as described earlier by Madhuprakash et al., (2015) ( 48 ). The resulting periplasmic fractions were dialyzed against the equilibration buffer (50 mM NaH 2 PO 4 , 300 mM NaCl and 10 mM imidazole, pH 8.0), prior to purification. The truncated variants, i.e. DUFA-DUFB, DUF-A, DUF-B and CBM73, were produced intracellularly and thus isolated by sonication of the cells, as follows: the cell pellets were resuspended in equilibration buffer and disrupted by pulsed sonication for 5 min on ice (cycles of 20 s on and 40 s off), and the cell debris were removed by centrifugation at 18,000 rpm for 45 min at 4°C. The supernatant obtained was filtered through a 0.45 µm membrane filters (Merck Millipore, USA) and purification was performed by Ni-NTA affinity chromatography as reported previously ( 49 ). Fractions with the highest purity were pooled together and concentrated by centrifugation at 3,200 rpm using Vivaspin® Turbo 15 Ultrafiltration Units of 10 kDa MWCO (Sartorius, Germany). The final concentrated protein fractions were subjected to buffer exchange against 50 mM sodium phosphate, pH 8.0. The protein concentration was estimated using Pierce BCA (Bicinchoninic acid) assay kit (Thermo Scientific, USA) as per the manufacturer’s protocol. The GH18 chitinases, Ec ChiA (uni-modular) and Ec ChiB (multi-modular) were also cloned from the genomic DNA of E. cloacae ATCC 13047 into pET-28a(+) and pET-22b(+) expression vectors, respectively, under the restriction sites, Nco I and Xho I (Thermo Scientific, USA). Sequence-validated clones were transformed by heat-shock into chemically competent BL21 (DE3) pLysS E. coli cells. Colonies obtained were inoculated into lysogenic broth (LB) containing 50 μg/mL kanamycin (in case of Ec ChiA) or 100 μg/mL ampicillin (in case of Ec ChiB). The recombinant chitinases were expressed by growing the transformants at 37°C for 3 h (until the OD reached 0.4-0.6) followed by induction with 0.4 mM IPTG for 16 h at 18°C and shaking at 180 rpm. The cells were harvested by centrifugation at 8000 rpm for 15 min. The cell pellet were resuspended in equilibration buffer containing 0.1 mM phenylmethylsulfonyl fluoride and sonicated using a Sonic-Ultrasonic Processor VCX-750 sonicator. The sonication was performed at 30% amplitude for 5 min at 20 s on and 40 s off cycles. Frequent monitoring of the sample was done to avoid heating. The sonicated sample was then centrifuged at 18000 rpm for 45 min at 4°C to separate the pellet and supernatant. The supernatant was purified by Ni-NTA affinity chromatography, followed by concentration and buffer exchange using Vivaspin® Turbo 15 Ultrafiltration Units of 10 kDa MWCO (Sartorius, Germany) as mentioned above. Protein estimation was done using Pierce BCA (Bicinchoninic acid) assay kit (Thermo Scientific, USA) as per the manufacturer’s protocol. 2.5. LPMO reaction and products analysis 2.5.1. MALDI-TOF mass spectrometric analysis of oxidized products The reaction mixture was composed of 1 μM of Ec LPMO FL , 10 mg/ml of α-or β -chitin (Stellar Biosol, Gujarat, India), 1 mM ascorbate, in 5 mM Tris-Cl pH 8.0. The reaction was incubated at 37°C for 24 h, at 1,000 rpm in an Eppendorf Thermomixer® C (Eppendorf India Pvt. Ltd., Chennai, India). Samples were collected at 12 h and 24 h and filtered using a 96-well filter plate (0.45 μm filters; Merck Millipore, USA) operated by a Millipore vacuum manifold. The filtered samples were lyophilized and analysed by MALDI-TOF-MS analysis as described previously ( 1 ). 2.5.2. Detection of H 2 O 2 production by Amplex red assay The apparent hydrogen peroxide generation during substrate-free Ec LPMO reactions was followed by using the Amplex Red/HRP assay as established by Kittl et al. (2012) ( 50 ). Briefly, a pre-reaction mixture contained (final concentrations after all additions): HRP (0.1 mg/mL), Amplex red (200 µM) and Ec LPMO FL , Ec LPMO CD , Ec LPMO ΔCBM73 or Apo- Ec LPMO ΔCBM73 (1 µM) in sodium phosphate buffer (pH 7.0, 50 mM). The final reaction (100 µL in a 96-well microplate), conducted at 25°C, was initiated by the addition of ascorbate (50 µM). The formation of resorufin (2:1 ratio with respect of H 2 O 2 ), was monitored at 575 nm over a 90-minute duration using a microplate reader (BioTek Epoch 2, Agilent technologies, US). The background of non-enzymatic H 2 O 2 production was assessed under identical conditions and no LPMO and was comparable to the reaction with the non-metalated LPMO (“Apo”). H 2 O 2 solutions, prepared in TraceSELECT water were used to generate a standard curve under the same conditions. The concentration of H 2 O 2 stock solution was verified at 240 nm (ε 240 = 43.6 M -1 .cm -1 ). 2.5.3. Product analysis using HPAEC-PAD Time course degradation experiments were performed by mixing (in a total reaction volume of 1 mL) 1 μM of enzyme ( Ec LPMO FL , Ec LPMO CD or Ec LPMO ΔCBM73 ) and 10 mg/mL of α-or β-chitin in 50 mM Tris-Cl pH 8.0. The reaction was initiated by addition of 1 mM ascorbate. The reaction was performed at 37°C and 1000 rpm for 24 h in an Eppendorf ThermoMixer C®. An aliquot of 150 μL was collected at different time points and filtered using a 96-well filter plate operated by a Millipore vacuum manifold and the filtrate was analyzed using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD, DIONEX ICS6000 system, Thermo Fisher Scientific, Waltham, MA, USA), as previously described ( 22 ). The system was equipped with a CarboPac-PA1 guard column (2 x 50 mm) and a CarboPac-PA1 column (2 x 250 mm) kept at 30°C. All reactions were performed in triplicate with appropriate control reactions under the similar experimental conditions as required. 2.6. Binding assay on chitin substrates The binding assays were conducted following the protocol previously described by Forsberg et al. (2016) ( 40 ) with slight modifications. Each reaction contained 10 mg/mL of α-or β-chitin substrates with 100 μg/mL of protein in 50 mM sodium phosphate buffer pH 7.0. The reaction was incubated for different time points (2.5, 5, 7.5, 10, 15, 30, 45 and 60 minutes) using an Eppendorf Thermomixer C® (Eppendorf India Pvt. Ltd., Chennai, India) at 1000 rpm. These reactions were performed at 22°C for CBM73 and at 4°C for the other truncations i.e., Ec LPMO FL , Ec LPMO CD and Ec LPMO ΔCBM73 . These temperatures were standardized based on the quality of binding isotherms observed for each truncated variant. Binding studies for the independent Ec LPMO domains, i.e., DUF-A+B, DUF-A and DUF-B on both α-and β-chitin were performed at 22°C at different time points (0, 6, 12, 24, 48 h for DUF-A+B double domain; 0, 15, 30, 45, 60, 90, 120, 180 min for DUF-A and DUF-B single domains). Binding affinities of these truncations were verified under different pH conditions, i.e., 50 mM sodium phosphate pH 8.0, 50 mM bis-tris pH 6.0 and 50 mM CAPS pH 10.0, keeping all other reaction parameters constant. Binding kinetics were performed to obtain the equilibrium binding constants ( K d ) and substrate binding capacity ( B max ) by using different concentrations of the protein (0, 10, 20, 50, 75, 100, 150, and 300 μg/mL) in 50 mM sodium phosphate pH 7.0 with 10 mg/mL α-or β-chitin. Firstly, a standard curve of the individual proteins was prepared in 50 mM sodium phosphate pH 7.0 without the substrate. In all conditions, i.e. in the presence and absence of the substrate, the reaction mixtures were incubated at 22°C for CBM73 and at 4°C in case of Ec LPMO FL , Ec LPMO CD and Ec LPMO ΔCBM73 for 2 h in an Eppendorf Thermomixer C® at 1,000 rpm. Post-incubation, the reaction mixtures were centrifuged at 11,000 rpm for 10 min at 4°C. The quantity of protein in the supernatant (referred to as “free protein”, P free ) was analyzed by the Pierce Bicinchoninic Acid (BCA) assay kit (Thermo Scientific, USA). All the assays were performed in triplicates with suitable controls. K d and B max were determined by fitting the binding isotherms to the one-site binding equation: [ P bound ] = B max [ P free ]/ K d +[ P free ], where P represents the protein, by non-linear regression using the GraphPad Prism software (GraphPad Software Inc., San Diego, CA). 2.7. Synergy experiments for chitin degradation Synergy experiments were performed by incubating 10 mg/mL of α-or β-chitin with 1 μM of Ec LPMO FL or its truncated variants ( Ec LPMO CD and Ec LPMO ΔCBM73 ), in combination with 1 μM of Ec ChiA or Ec ChiB, in 50 mM Tris-Cl pH 8.0, in the presence of 1 mM ascorbate. All the reactions were performed in triplicate, and incubated at 40°C, 1,000 rpm in an Eppendorf ThermoMixer C®, up to 24 h. Aliquots were collected at different time points and filtered using a 96-well filter plate (0.45 μm filters; Merck Millipore) operated by a Millipore vacuum manifold. The filtrates were mixed with equal volume of 70% acetonitrile and analyzed by HPLC on the Shim-pack GIST NH2 column (4.6 mm ID X 250 mm, Shimadzu, Japan) through isocratic elution using 70% acetonitrile at a flow rate of 0.7 mL/min. CHOS were detected by monitoring the absorbance at 210 nm. Quantification of the neutral chitooligosaccharides (CHOS) generated was performed essentially as described previously ( 51 , 52 ). 3. Results 3.1. Bioinformatic analyses of EcLPMO To get preliminary insights into Ec LPMO properties and function, we carried out sequence and phylogenetic analyses. Ec LPMO FL is a multi-modular LPMO comprising a total of 484 amino acids. It includes a 21-residue N-terminal signal peptide, followed by an AA10 catalytic domain (residues 22-190), two domains of unknown function DUF-A (residues 205-308) and DUF-B (residues 316-413) and a C-terminal CBM73 domain (residues 424-484) ( Fig. 1A and B ) . A phylogenetic analysis of the catalytic domain only of 176 AA10 LPMOs showed that Ec AA10 belongs to a clade of well-known chitin-active AA10s ( Fig. 1C ). In details, Ec AA10 shares a sequence identity of 51.5%, 48.8%, and 33.5% with the AA10 catalytic domains of GbpA (PDB: 2XWX), Vca LPMO10A (PDB: 8GUM), and CbpD (PDB: 7SQX), respectively, which are all chitin-active ‘GbpA-type’ LPMOs ( 27 , 32 , 35 ). Multiple sequence alignment revealed the conservation of the ‘histidine brace’ residues (His1 and His88 in Ec LPMO mature sequence (i.e. without SP); Fig. S1 ). Download figure Open in new tab Fig. 1. Sequence analysis of Ec LPMO. (A) Domain architecture of full-length Ec LPMO ( Ec LPMO FL ) and its truncated variants. The constructs, including Ec LPMO FL , Ec LPMO ΔCBM73 , and Ec LPMO CD , contain a predicted signal peptide (SP; residues 1–22). (B) AlphaFold3-predicted structure of Ec LPMO FL , highlighting its modular domain organization. (C) Phylogenetic tree of the catalytic domains from 176 AA10 LPMOs. Bootstrap values are shown at branch nodes as blue circles (see scale in figure). Red filled stars mark previously characterized tetra-modular LPMOs orthologs of Ec LPMO (printed in red). Regarding the DUF domains, DUF-A shares sequence identities of 40%, 37.6% and 34% with the GbpA D2 (GbpA_2 domain), the Vca LPMO10A-GbpA_2 and CbpD-MX (GbpA_2 domain), respectively. Meanwhile, DUF-B has a relatively low sequence identity of 30.1% and 24.7% compared to GbpA D3 and Vca LPMO10A-MX, respectively. To evaluate the evolutionary relationship of the CBM73 domain with other members of the CBM73 family, we performed a phylogenetic analysis using 572 representative sequences clustered at 90% identity from 5,025 CBM73 entries available in the CAZy database. The resulting phylogenetic tree revealed five major clades, which likely reflect underlying functional divergence within the family ( Fig. 2A ). Notably, the CBM73 family remains largely uncharacterized, with most clades lacking any biochemically or structurally studied members, likely due to its relatively recent identification ( 40 ). Interestingly, approximately half of the representative sequences fall within a single superclade (highlighted in purple), which can be further resolved into multiple subclades. Ec CBM73 clusters within this purple superclade, alongside two previously characterized CBM73s, Pa CBM73 ( 42 ) and Vca CBM73 ( 35 ), though each resides in a separate subclade. Ec CBM73 shares only moderate sequence identity with these known homologs: 33.3% with Cj CBM73, 49.2% with Pa CBM73, and 60.3% with Vca CBM73. Structural superimposition of Ec CBM73 model (AF3) with X-ray crystallographic structures of Pa CBM73 and Vca CBM73 shows high similarity (RMSD values of 0.298 Å and 0.45 Å for C α atoms, respectively). In contrast, superposition with Cj CBM73 revealed more pronounced structural differences, with an RMSD of 1.508 Å, supporting the phylogenetic separation of these CBMs. Download figure Open in new tab Fig. 2: Phylogenetic tree of the CBM73 domain. (A) The tree was built with the CBM73 domain of 572 sequences, representative (on the basis of sequence clustering with 90% identity threshold) of 5,025 sequences annotated in the CAZy database. The tree was inferred using IQ-Tree ( 52 ) (1,000 ultrafast bootstraps; values displayed on the tree) and visualized with the Interactive Tree Of Life (iTOL) software ( 45 ). Characterized CBM73s, namely from Pseudomonas aeruginosa PA01 (GenBank ID AAG04241.1) ( 42 ), from Cellvibrio japonicus Ueda107 (GenBank ID ACE83992.1) ( 43 ), and from Vibrio campbellii ATCC BAA-1116 (GenBank ID ABU72648.1) ( 35 ) are highlighted on the tree. (B) Substrate-binding surface view highlighting solvent-exposed residues (shown as sticks) of, from top to bottom, Ec CBM73 (AF model), Vca CBM73 (PDB 8GUM; 35), Pa CBM73 (AF model) and Cj CBM73 (AF model). (C) WebLogo of the MSA of subclades (delimited by grey lines in panel A) containing Ec CBM73, Pa CBM73 and Vca CBM73, and corresponding to the stretch of amino acids forming the substrate binding surface (shown in panel B). Residues not highlighted in panel B are masked by a grey shading in panel C. The same numbering of highlighted residues has been used in both panels B and C. Note that some residues shown in panel B have a very low frequency of occurrence within the subclade (e.g. Trp at pos. 6 in Pa CBM73), and are thus barely readable in the corresponding WebLogo shown in panel C. 3.2. Comparison of CBM73 substrate-binding surfaces and sequence features To further investigate the potential functional implications of these evolutionary relationships, the substrate-binding surfaces of Ec CBM73, Vca CBM73, Pa CBM73, and Cj CBM73 were compared based on their modelled structures ( Fig. 2B ). The three CBM73s from the purple clade ( Ec CBM73, Vca CBM73, and Pa CBM73) exhibit more extended binding surfaces relative to Cj CBM73, which presents a compact and shallow architecture. Among the extended forms, Ec CBM73 displays the broadest surface (∼31.1 Å), followed by Vca CBM73 (∼28.7 Å) and Pa CBM73 (∼21.3 Å), while Cj CBM73 exhibits the narrowest (∼14.2 Å). These differences likely reflect clade-specific substrate recognition strategies and may point to distinctions in binding affinity. Sequence logos derived from each CBM73 subclade ( Fig. 2C ) reveal partial conservation at key positions, such as a highly conserved proline at position 2, likely important for maintaining the core structural framework. However, other regions exhibit notable divergence. For instance, comparing Ec CBM73 to Vca CBM73 reveals substitutions at position 3 (tyrosine vs. phenylalanine), potentially introducing additional polar interactions or affecting aromatic stacking at the substrate interface. Similarly, at position 7, Ec CBM73 harbours a tyrosine residue, whereas Vca CBM73 features a serine, representing a shift from an aromatic to a polar side chain that could influence substrate interactions through altered hydrogen bonding or stacking potential. Interestingly, in Pa CBM73, residues corresponding to positions 7 and 8 are entirely absent, resulting in a truncated loop that may contribute to its comparatively narrower binding surface. Such variations, particularly in loop length and residue chemistry, underscore the structural plasticity and potential functional diversification of CBM73s within this clade. Additional substitutions at other positions (e.g., 10 and 12) further emphasize the evolutionary dynamics shaping this domain family. Together, these structural and sequence-level observations support the idea that CBM73s, particularly those within the same clade, retain a conserved fold but adaptively diverge in surface architecture and residue composition. These variations may allow fine-tuning of binding properties to specific polysaccharide substrates or structural contexts in the broader LPMO scaffold. 3.3. EcLPMO is a functional chitinolytic LPMO In order to probe the activity of Ec LPMO, Ec LPMO FL and the six truncations thereof were successfully produced in E. coli , purified by IMAC, and submitted to several biochemical assays. First, using MALDI-TOF-MS, we verified that Ec LPMO FL , in the presence of an electron donor (ascorbate), oxidatively cleave α- and β-chitin, as shown by the release of oxidized chitooligosaccharides (CHOS) with degree of polymerization (DP) ranging from DP2 to DP10 ( Fig. S2 ). The presence of single and double sodium adducts, [M+Na] + and [M– H+2Na] + , is characteristic of C1-oxidized CHOS (aldonic acids), consistent with previous reports ( 1 ) ( Fig. S2 ). Notably, Ec LPMO FL preferentially released even-chain oxidized oligosaccharides from both α- and β-chitin. Monitoring the release of oxidized CHOS revealed that the full-length enzyme Ec LPMO FL was more efficient than Ec LPMO CD and Ec LPMO ΔCBM73 over the entire time-course, on both α- and β-chitin ( Fig. S3 ). Intriguingly, removal of the CBM73 only nearly abolished the activity, while the Ec LPMO CD maintained an intermediate activity. These observations suggest that the DUF domains may play a role in the mode of action of the LPMO on polysaccharides (see more below and in the Discussion ). In addition, the Amplex red assay showed that, in the presence of ascorbate, Ec LPMO FL reduces O 2 into H 2 O 2 ( Fig. S4) , at a rate comparable to previously characterized LPMOs ( 15 ). Experiments with Ec LPMO CD and Ec LPMO ΔCBM73 led to similar values (2-3 x 10 -3 s -1 ), indicating that, under those conditions, the accessory modules do not affect the reactivity at the copper site (see more in Discussion ). Of note, control experiments with the apo enzyme showed only a low residual activity. 3.4. EcCBM73 is crucial for efficient chitin binding Binding assays were performed with Ec LPMO FL , its truncated variants ( Ec LPMO ΔCBM73 and Ec LPMO CD ), and the isolated Ec CBM73 domain to assess their affinity for α- and β-chitin ( Fig. 3A and B ) . Ec CBM73 exhibited the strongest binding, reaching 89.3% and 86.9% binding to α- and β-chitin, respectively, within just 10 minutes ( Fig. 3A and B ) . In comparison, Ec LPMO FL showed slower binding kinetics, with maximal binding of approximately 55% to both substrates after 60 minutes ( Fig. 3A and B ) . The truncated variants displayed notably weaker binding: Ec LPMO ΔCBM73 and Ec LPMO CD reached only 38–41% binding to α-chitin ( Fig. 3A ) and 43–57% to β-chitin ( Fig. 3B ) . These results underscore the significant contribution of the CBM73 domain to both the binding kinetics and binding capacity of the full-length enzyme. Download figure Open in new tab Fig. 3: Binding studies of Ec LPMO FL and its truncated variants on α- and β-chitin. (A) and (B) Represent the time-course binding of Ec LPMO FL and its truncated variants to α- and β-chitin, respectively. The percentage of free protein was determined by measuring the protein concentration in the reaction supernatant collected over time using the BCA method. The experiments were carried out at 22°C using 10 mg/mL substrate in 50 mM sodium phosphate buffer, pH 7.0. (C) and (D) Represent comparison of K d (µM) and B max (µmol/g chitin), respectively, for Ec LPMO FL and its truncated variants, using α- and β-chitin as substrate. Data points (values given in Table S4 ) show average values and error bars show standard deviations (n = 3 independent biological replicates). In panels (C) and (D), Ec LPMO FL and its truncated variants, Ec LPMO ΔCBM73 , Ec LPMO CD and Ec CBM73 have been denoted as FL, ΔCBM73, CD and CBM73, respectively. To further examine the potential role of other regions of the protein, binding studies were also conducted for the double domain DUF-A+B, and the single domains DUF-A, and DUF-B, on both chitin forms under varying pH conditions. At pH 8.0 and 9.0, none of these domains exhibited appreciable binding to either substrate ( Fig. S5 ). However, at pH 10, DUF showed minimal binding (20–24% for both α- and β-chitin; Fig. S5A, B ), while DUF-A displayed moderate binding, up to 49% to α-chitin and 35% to β-chitin ( Fig. S5C, D ). DUF-B showed no significant binding under any tested condition ( Fig. S5E, F ). To gain quantitative insights into the binding behaviour, binding isotherms were generated by varying protein concentration at fixed chitin loading, allowing determination of B max and K d values ( Fig. 3C and D ; Table S4 ). A clear trend emerged: progressive truncation of Ec LPMO FL ( Ec LPMO FL → Ec LPMO ΔCBM73 → Ec LPMO CD ) led to increasing divergence in substrate preference between α- and β-chitin. Notably, both Ec LPMO FL and Ec CBM73 alone showed comparable affinity for α- and β-chitin (based on K d and B max ), whereas Ec LPMO ΔCBM73 and Ec LPMO CD exhibited a substantial reduction in affinity (higher K d ) for β - chitin, alongside displaying an expected reduction in B max relative to the full-length enzyme ( Fig. 3D ; Table S4 ). On α-chitin as well, the truncated variants exhibited high K d values, indicating reduced substrate affinity, while their B max values were comparable to or slightly higher than those of Ec LPMO FL ( Fig. 3C ; Table S4 ). Indeed, truncation of CBM73 or the removal of additional domains led to a marked loss in substrate affinity, particularly for β-chitin, indicating that these accessory regions modulate substrate preference. Interestingly, despite this reduction in affinity, the truncated variants displayed moderately elevated B max values on α-chitin. This apparent paradox, poorer affinity but higher maximal binding, likely reflects increased exposure or conformational flexibility of the catalytic domain in the truncated forms, allowing more enzyme molecules to interact non-specifically or transiently with the substrate. These findings suggest that in the Ec LPMO FL , the accessory modules, especially CBM73, may restrict the number of catalytically engaged enzymes but confer higher binding specificity and stability. Moreover, the progressive loss of selectivity between α- and β-chitin upon domain deletion implies a possible structural tuning of the full-length enzyme towards substrates that resemble α-chitin more closely. Such domain-mediated modulation of substrate interaction may be critical for function in physiological contexts and highlights the sophisticated role of modularity in fine-tuning LPMO activity. 3.5. EcLPMO FL boosts the activity of E. cloacae chitinases To assess the potential interplay between E. cloacae chitinases, specifically Ec ChiA, a unimodular GH18 enzyme ( 53 ), and Ec ChiB, a multimodular enzyme comprising an N-terminal GH18 catalytic domain, a polycystic kidney disease (PKD) domain, and two tandem chitin-binding modules ( 54 ), and Ec LPMO FL , we performed synergy assays using both α- and β-chitin substrates. The addition of Ec LPMO FL substantially enhanced the activity of both chitinases on α-chitin ( Fig. 4A, B ) and β-chitin ( Fig. 4C, D ). Notably, Ec LPMO FL displayed a particularly strong synergistic effect with Ec ChiA, resulting in up to 14-fold and 60-fold increases in GlcNAc release from α- and β-chitin, respectively, compared to reactions containing the chitinase alone ( Fig. 4A, C ). Synergy with Ec ChiB was also evident, though less pronounced, yielding up to 2-fold and 1.6-fold increases in (GlcNAc) 2 release from α- and β-chitin, respectively ( Fig. 4B, D ). Given the pronounced synergistic enhancement observed with Ec ChiA, this chitinase was selected for subsequent experiments involving truncated Ec LPMO variants. Download figure Open in new tab Fig. 4: Synergistic chitin degradation by Ec LPMO FL and E. cloacae chitinases. The graphs show the time-course release of total soluble GlcNAc and (GlcNAc) 2 by E. cloacae chitinases ( Ec ChiA or Ec ChiB; 1 µM), in the absence or presence of Ec LPMO FL (1 µM) and ascorbate (1 mM), during the degradation of α-chitin (A and B) and β-chitin (C and D). Data points show average values and error bars show standard deviations (n = 3 independent biological replicates). 3.6. Deletion of EcCBM73 impairs the synergistic activity of EcLPMO with chitinases To evaluate the contribution of Ec LPMO FL modular structure to its synergistic function with Ec ChiA, we compared the full-length enzyme with its truncated variants, Ec LPMO ΔCBM73 and Ec LPMO CD . Overall, in contrast to Ec LPMO FL , both truncated variants showed markedly reduced performance on both α- and β-chitin ( Fig. 5A ). More in details, on α-chitin, the truncated variants resulted in nearly a two-fold reduction in GlcNAc yield at 24 h compared to the full-length enzyme ( Fig. 5A ). Similarly, (GlcNAc) 2 yields dropped by ∼2.9-fold and ∼2.6-fold for Ec LPMO ΔCBM73 and Ec LPMO CD , respectively ( Fig. 5B ). For β-chitin, GlcNAc release decreased by up to 5.5-fold in reactions with Ec LPMO ΔCBM73 , while no detectable products were observed with Ec LPMO CD beyond the 24 h mark ( Fig. 5C ). This trend was mirrored in (GlcNAc) 2 release profiles ( Fig. 5D ). Together, these findings highlight the essential role of the CBM73 domain in sustaining and enhancing the synergistic action of Ec LPMO FL with chitinases. Download figure Open in new tab Fig. 5: Synergistic effect of Ec LPMO FL and its truncated variants on crystalline chitin degradation by Ec ChiA. The graphs show the time-course release of total soluble GlcNAc and (GlcNAc) 2 by Ec ChiA (1 µM), in the absence or presence of Ec LPMO FL and its truncations ( Ec LPMO ΔCBM73 and Ec LPMO CD ) (1 µM) and ascorbate (1 mM), during the degradation of α-chitin (A and B) and β-chitin (C and D). Data points show average values and error bars show standard deviations (n = 3 independent biological replicates). 3.7. The chitinolytic machinery of Enterobacter cloacae In an attempt to draw a broader picture of Ec LPMO function, we examined its genomic context, unveiling an elaborate chitinolytic system comprising two GH18 chitinases, two GH19 chitinases, two β -N-acetylhexosaminidases (GH3 and GH20), and one CE4 polysaccharide deacetylase. Several of these enzymes are genomically co-localized, indicating potential functional associations ( Fig. 6 ). Ec LPMO is located in the same operon as Ec HexA (GH20), while Ec ChiB and Ec ChiC are adjacent to a predicted type II secretion system ( Fig. 6A ). The remaining genes are dispersed. Among the encoded chitin-active CAZymes, Ec ChiA, Ec ChiB, Ec ChiC, Ec HexA and Ec LPMO, possessed an N-terminal signal peptide, indicating extracellular secretion ( Fig. 6B ). An illustration of chitin degradation mediated using these secreted enzymes by E. cloacae have been represented in Fig. 6C . This chitinolytic gene repertoire is conserved across E. cloacae strains, with Ec LPMO among the most conserved components ( Table S2 ). Download figure Open in new tab Fig. 6: Schematic representation of extracellular chitin degradation by E. cloacae . (A) Represents the genetic loci depicting gene arrangement of the chitin-active CAZymes of E. cloacae ATCC 13047 T that possess an N-terminal signal peptide, indicating extracellular secretion. The direction of the arrows indicates the strand on which they are located. Genes with overlapping loci are indicated with arrows outside the frame. The size of the arrows represents the gene size (scale in base pairs provided at the bottom of the figure). The arrows color code is as follows: red, genes coding for the chitin-active CAZymes (the label within the arrow indicates the CAZyme family, while the one at the bottom represents the name of the gene/protein); yellow, genes involved in the general secretory pathway; grey, genes encoding hypothetical proteins; black, unrelated genes. (B) Represents the domain architecture of these chitin-active CAZymes. Abbreviations: SP – Signal Peptide; GH – Glycoside Hydrolase; Hex-β-N-acetylhexosaminidase; PKD – Polycystic Kidney Disease domain; CBM – Carbohydrate Binding Module; GH20b – N-terminal domain of GH20; DUF – Domain of Unknown Function. (C) Represents an illustration of chitin degradation mediated by E. cloacae using the secreted enzymes shown in (A and B). Note: Ec ChiC and Ec HexA are yet to be characterized and hence their representation was made based on CAZy database prediction and existing literature. 4. Discussion LPMOs are a unique class of enzymes originally identified for their critical role in the oxidative cleavage of polysaccharides during biomass degradation. However, emerging studies have expanded their functional scope ( 26 ), revealing involvement in other biological contexts, including their roles as virulence factors in bacterial pathogenesis. These include AA10 LPMOs from human pathogens – viz., the Lm LPMO10 from Listeria monocytogenes ( 55 , 56 ), GbpA from Vibrio cholerae ( 32 , 57 , 58 ), and CbpD from Pseudomonas aeruginosa ( 27 ), along with proteins from pathogens affecting animals, such as fusolin from Entomopoxvirus ( 19 ), Pl CBP49 from Paenibacillus larvae ( 59 ), and As LPMO10A/B from Aliivibrio salmonicida ( 60 ). These findings have broadened the understanding of LPMO function across diverse pathogenic bacteria. In this study, we examined an AA10 LPMO from an important bacterial human pathogen, namely E. cloacae . Interestingly, analysis of the available genomes of E. cloacae strains revealed that most strains (40 out of 46) encode a single lpmo gene ( Table S1 ), suggesting strong conservation and possible functional indispensability of this gene. Ec LPMO is a GbpA-type LPMO with a modular organization resembling GbpA from V. cholerae ( 32 ), CbpD from P. aeruginosa ( 27 ), and Vca LPMO10A from V. campbellii ( 35 ). We showed here that Ec LPMO, alike the latter LPMOs, can bind and oxidize crystalline chitin. Moreover, it showed notable synergy with the GH18 chitinase Ec ChiA, enhancing chitin breakdown to GlcNAc. In contrast, co-incubation with the multi-modular Ec ChiB primarily yielded (GlcNAc) 2 , highlighting differential synergy between Ec LPMO and chitinases. Diving further into the boosting effect of Ec LPMO, we found that the CBM73 plays a crucial role as shown by the reduced chitin degradation capacity of Ec LPMO ΔCBM73 and Ec LPMO CD variants and their lack of synergy with Ec ChiA. The strong chitin-binding properties of Ec CBM73, similar to its homolog in other bacteria ( 27 , 40 ) albeit with some differences in binding strength, probably explain its important role in sustaining Ec LPMO function. It has indeed been shown that productive polysaccharide binding is key to prevent LPMO oxidative auto-inactivation and thus maximize its boosting potential ( 11 , 61 , 62 ). Despite several studies, including the present one, on multi-modular LPMOs from pathogenic bacteria, the role of the DUF domains in multimodular LPMOs has remained hitherto mysterious. We have shown here that the DUF-A and DUF-B domains have very limited chitin-binding properties ( Fig. S5 ). During the preparation of the present manuscript, the release of AF3 software ( 46 ) has allowed to generate new models in the presence of copper, which revealed an unprecedented observation: a unique inter-domain arrangement in which the DUF-B domain folds onto the catalytic AA10 domain, forming an extended metal coordination environment composed of four histidine residues ( Fig. 7 , Fig. S6 ). In this configuration, two histidines from the AA10 domain coordinate the catalytic copper ion, while two additional histidines from the DUF-B domain complete a predicted tetra-histidine coordination sphere. This predicted inter-domain rearrangement is lost upon mutation of the histidine residues from the DUF-B domain (H354A and H384A in Ec LPMO; H357A and H388A in Vc GbpA), suggesting that both these residues and Cu(II) are essential for its formation. Notably, confidence in the relative positioning of DUF-A and DUF-B increases markedly when Cu(II) is included during modelling, as reflected in the PAE data for both proteins, further underscoring the potential role of copper in stabilizing the overall tertiary structure ( Fig. 7 ). Download figure Open in new tab Fig. 7: AlphaFold3-predicted structures of mature Ec LPMO and Vc GbpA including wild-type and His-to-Ala mutants, modelled alone or in complex with Cu(II). (A) (i-iv) Cartoon representations of AlphaFold3 (AF3) models are shown for wild-type mature Ec LPMO and the double mutant H354A, H384A , each modelled with and without Cu(II). Domains are shaded based on InterProScan annotations: LPMO (yellow), DUF-A (red), and DUF-B (green). Insets highlight the histidine brace region, with histidine side chains rendered as sticks and colored in red. Corresponding Predicted Alignment Error (PAE) plots are shown for each model, alongside the pTM and ipTM confidence metrics. All models were generated using AlphaFold3 with fixed seed number 1. (B) (i-iv) AF3 Models of wild-type mature Vc GbpA and the H357A, H388A mutant, with and without Cu(II), are shown using the same visual scheme. The analysis of structural models from other multi-modular LPMOs suggests a conservation of this tetra-histidine copper-coordinated center ( Fig. S6) . This configuration is, to our knowledge, being reported for the first time and represents a previously unrecognized feature of multi-modular LPMOs. Given the known susceptibility of LPMO active sites to oxidative self-inactivation (11, 61, 63-65), particularly under uncoupled turnover conditions where reactive oxygen species may accumulate, this tetra-histidine arrangement may reflect an evolutionary adaptation to protect the catalytically essential histidine brace from oxidative damage. Thus, in complement to substrate binding (11, 61, 63-65), and second sphere aromatic residues ( 66 , 67 ), we propose that such unique conformation may represent a new protective strategy. Such configuration could be a way for the microorganism to control the LPMO activity, by for instance rendering the AA10 domain functional upon unfolding of the multi-modular structure only upon binding of its proper substrate. Both hypotheses await, however, to be experimentally tested. In this context, the strong chitin-binding affinity of CBM73 may serve a dual role beyond substrate recruitment. Based on our results with Ec LPMO truncation variants, where Ec LPMO ΔCBM73 displayed minimal or no activity while Ec LPMO CD retained measurable activity, it is tempting to propose that CBM73-mediated substrate engagement facilitates a conformational unlocking of the catalytic site. Specifically, substrate binding may induce structural rearrangement that destabilizes or ‘unshields’ the DUF-B domain from its folded position over the histidine brace, transitioning the enzyme from a protected, inactive state to an open, catalytically competent form. Furthermore, the redox state of the copper might also play an important role in the ‘unshielding’ process. Indeed, the fact that the full-length and truncated enzymes produce H 2 O 2 at similar rates ( Fig. S4 ) suggests that the copper is equally accessible, and, thus, that the tetra-modular copper coordination conformation is not formed under the assay’s reducing conditions. This resonates with previous observations: in its Cu(II) state, the copper ion bound to single AA10 LPMO domains typically coordinates (in addition to the His-Brace) two water molecules ( 68 ), which are presumably replaced here by the two DUF-B histidines. Upon reduction of Cu(II) to Cu(I), a coordination site is lost in single LPMO domains (one water molecule is excluded; 68), which may, here, be translated in the disruption of the tetra-histidine coordination, explaining thereby the similar Cu(I) reactivity between WT and truncated enzymes. These possibilities, though speculative, offer compelling directions for future structural and biochemical investigation. Taking into account all data presented in this study, we propose that the chitinolytic machinery of E. cloacae may aid nutrient acquisition in chitin-rich environments such as soil. Alternatively, or in complement, the relevance of these enzymes during host colonization or infection remains to be explored. Indeed, among Ec LPMO homologs, GbpA from V. cholerae has been studied for its effects on host cells, including mitochondrial dysfunction, ROS production, and NF-κB activation, leading to necrotic cell death ( 69 ). In contrast, CbpD from P. aeruginosa contributes to immune evasion by inhibiting complement activation and resisting the membrane attack complex ( 27 ). Although Ec LPMO shares domain architecture with these proteins, its low sequence similarity suggests divergent functions. Nevertheless, its strong conservation across E. cloacae strains implies a potentially important role in host-pathogen interactions. 5. Conclusion In summary, Ec LPMO represents a distinct multi-modular LPMO with chitin-binding and oxidative activity, functioning in synergy with endogenous chitinases. Importantly, the observations made on the individual roles of each module, and notably the presence of putative inter-domain tetra-histidine copper coordination center, expand our understanding of LPMO structural diversity and suggest potential avenues for engineering LPMOs with enhanced oxidative resilience and stability. Furthermore, its conserved presence across E. cloacae strains, along with its modular similarities to known virulence-associated LPMOs, points to a potentially broader biological role, including in host-pathogen interactions. These findings highlight Ec LPMO as a promising candidate for further structural and functional studies, both in the context of microbial pathogenesis and environmental chitin turnover. Funding Support The authors would like to express their gratitude to the Department of Science and Technology (DST), Government of India (GoI), for the Funds for Infrastructure in Science and Technology (FIST), Level III to the Department of Plant Sciences, UoH. The authors would also like to thank the Department of Biotechnology (DBT) for the DBT-SAHAJ/BUILDER, (BT/INF/22/SP41176/2020) support to the School of Life Sciences, UoH. Furthermore, JM would like to acknowledge the funding received from the DBT (BT/PR42225/BCE/8/1586/2021), Science and Engineering Research Board, GoI (CRG/2019/006426), and the UoH-Institution of Eminence (RC1-20-020). B.B. thanks INRAE (notably through the EvoFun project PAF_02) for supporting the research of this work. Acknowledgements We are grateful to Prof. Appa Rao Podile (Department of Plant Sciences, School of Life Sciences, University of Hyderabad) for kindly providing the genomic DNA of E. cloacae 13047 T . We also thank Prof. Bernard Henrissat and the CAZy team in Marseille, France, for their support in annotating the domain boundaries, specifically AA10, DUF-A, DUF-B, and CBM73. Funder Information Declared Department of Biotechnology, https://ror.org/03tjsyq23 , DBT-SAHAJ/BUILDER, (BT/INF/22/SP41176/2020) , BT/PR42225/BCE/8/1586/2021 Department of Science and Technology, https://ror.org/0101xrq71 , Funds for Infrastructure in Science and Technology (FIST), Level III to the Department of Plant Sciences, UoH Science and Engineering Research Board, https://ror.org/03ffdsr55 , CRG/2019/006426 UoH-Institution of Eminence , RC1-20-020 INRAE , EvoFun project PAF_02 References 1. ↵ Vaaje-Kolstad , G. , Westereng , B. , Horn , S. J. , Liu , Z. , Zhai , H. , Sørlie , M. , et al. ( 2010 ) An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides . Science 330 , 219 – 222 . OpenUrl Abstract / FREE Full Text 2. ↵ Quinlan , R. J. , Sweeney , M. D. , Leggio , L. L. , Otten , H. , Poulsen , J. C. N. , Johansen , K. S. , et al. ( 2011 ) Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components . 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