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. ### Competing Interest Statement The authors have declared no competing interest. 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

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 lytic polysaccharide monooxygenase (LPMO), termed EcLPMO, which we functionally characterized in this study. EcLPMO 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 EcLPMO 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. EcLPMO exhibited synergy with the unimodular chitinase EcChiA, resulting in up to 14-fold and 60-fold increase in GlcNAc release from α- and β-chitin, respectively. Deletion of both DUFs reduced EcLPMO 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. Using 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 EcLPMO 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.

Lytic polysaccharide monooxygenases (LPMOs) have an essential role in global carbon cycle, industrial biomass processing and microbial pathogenicity by catalysing the oxidative cleavage of recalcitrant polysaccharides. Despite initially being considered monooxygenases, experimental and theoretical studies show that LPMOs are essentially peroxygenases, using a single copper ion and H 2 O 2 for C–H bond oxygenation. Here, we examine LPMO catalysis, emphasizing key studies that have shaped our comprehension of their function, and address side and competing reactions that have partially obscured our understanding. Then, we compare this novel copper–peroxygenase reaction with reactions catalysed by haem iron enzymes, highlighting the different chemistries at play. We conclude by addressing some open questions surrounding LPMO catalysis, including the importance of peroxygenase and monooxygenase reactions in biological contexts, how LPMOs modulate copper site reactivity and potential protective mechanisms against oxidative damage.

In this paper, we employed a multidisciplinary approach, combining experimental techniques and density functional theory (DFT) calculations to elucidate key features of the copper coordination environment of the bacterial lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens (SmAA10). The structure of the holo-enzyme was successfully obtained by X-ray crystallography. We then determined the copper(II) binding affinity using competing ligands and observed that the affinity of the histidine brace ligands for copper is significantly higher than previously described. UV-vis, advanced electron paramagnetic resonance (EPR), and X-ray absorption spectroscopy (XAS) techniques, including high-energy resolution fluorescence detected (HERFD) XAS, were further used to gain insight into the copper environment in both the Cu(II) and Cu(I) redox states. The experimental data were successfully rationalized by DFT models, offering valuable information on the electronic structure and coordination geometry of the copper center. Finally, the Cu(II)/Cu(I) redox potential was determined using two different methods at ca. 350 mV vs NHE and rationalized by DFT calculations. This integrated approach not only advances our knowledge of the active site properties of SmAA10 but also establishes a robust framework for future studies of similar enzymatic systems.

The worldwide accumulation of plastic waste in the environment, along with its lifespan of hundreds of years, represents a serious threat to ecosystems. Enzymatic recycling of plastic waste offers a promising solution, but the high chemical inertness and hydrophobicity of plastics pose several challenges to enzymes. In nature, lytic polysaccharide monooxygenases (LPMOs) can act at the surface of recalcitrant biopolymers, taking advantage of their solvent-exposed active sites and appended carbohydrate-binding modules (CBMs). LPMOs can disrupt the densely packed chains of polysaccharides (e.g., cellulose) by the oxidation of C-H bonds. Given the similarities between these natural and artificial polymers, we aimed here at promoting plastic-binding properties to LPMOs, by swapping their CBM with three natural, surface-active accessory modules displaying different amphipathic properties. The polymer binding capacity of the resulting LPMO chimeras was assessed on a library of synthetic polymers, including polyester, polyamide, and polyolefin substrates. We demonstrated that the plastic binding properties of these engineered LPMOs are polymer-dependent and can be tuned by playing on the nature of the accessory module and reaction conditions. Remarkably, we gained full binding for some chimera LPMOs with striking results for polyhydroxyalkanoates (PHA). In the long term perspective of harnessing the unique copper chemistry of LPMOs to degrade plastics, we also provided the first evidence of LPMO-dependent modification of the PHA polymer, as supported by enzyme assays, gel permeation chromatography, and scanning electron microscopy. Altogether, our study provides the first roadmap for engineering plastic-binding ability in LPMOs, constituting a crucial first step on the evolutionary path toward efficient interfacial catalysis of plastic-active enzymes.

The angiogenin protein (ANG) is one of the most potent endogenous angiogenic factors. In this work we characterized by means of potentiometric, spectroscopic and voltammetric techniques, the copper complex species formed with peptide fragments derived from the N-terminal domain of the protein, encompassing the sequence 1-17 and having free amino, Ang1-17, or acetylated N-terminus group, AcAng1-17, so to explore the role of amino group in metal binding and cellular copper uptake. The obtained data show that amino group is the main copper anchoring site for Ang1-17. The affinity constant values, metal coordination geometry and complexes redox-potentials strongly depend, for both peptides, on the number of copper equivalents added. Confocal laser scanning microscope analysis on neuroblastoma cells showed that in the presence of one equivalent of copper ion, the free amino Ang1-17 increases cellular copper uptake while the acetylated AcAng1-17 strongly decreases the intracellular metal level. The activity of peptides was also compared to that of the protein normally present in the plasma (wtANG) as well as to the recombinant form (rANG) most commonly used in literature experiments. The two protein isoforms bind copper ions but with a different coordination environment. Confocal laser scanning microscope data showed that the wtANG induces a strong increase in intracellular copper compared to control while the rANG decreases the copper signal inside cells. These data demonstrate the relevance of copper complexes' geometry to modulate peptides' activity and show that wtANG, normally present in the plasma, can affect cellular copper uptake.

Lytic polysaccharide monooxygenases (LPMOs) are copper‐dependent enzymes involved in the degradation of recalcitrant polysaccharides such as cellulose or chitin. LPMOs act in synergy with glycoside hydrolases such as cellulases and chitinases by oxidatively cleaving a number of glycosidic bonds at the surface of their crystalline substrate(s). Besides their role in biomass degradation, some bacterial LPMOs have been found to be virulence factors in some human and insect pathogens. Photorhabdus luminescens is a nematode symbiont bacterium that is pathogenic to a wide range of insects. A single gene encoding a LPMO is found in its genome. In this work, we report the characterization of this LPMO, referred to as PlAA10. Surprisingly, PlAA10 lacks the conserved alanine residue (substituted by an isoleucine) found in the second coordination sphere of the copper‐active site in bacterial LPMOs. PlAA10 was found to be catalytically active on both α‐ and β‐chitin, and exhibits a C1‐oxidation regiospecificity, similarly to other chitin‐active LPMOs. The 1.6 Å X‐ray crystal structure confirmed that PlAA10 adopts the canonical immunoglobulin‐like fold typical for LPMOs. The geometry of the copper‐active site is not affected by the nearby isoleucine, as also supported by electron paramagnetic resonance. Nevertheless, the bulkier side chain of isoleucine protrudes from the substrate‐binding surface. A bioinformatic study on putative bacterial LPMOs unveiled that they exhibit some variability at the conserved active‐site alanine position with a substitution in about 15% of all sequences analyzed.DatabaseStructural data (atomic coordinates and structure factors) reported for PlAA10 are available in the Protein Data Bank under accession number 6T5Z.EnzymesPlAA10, EC1.14.99.53.

Angiogenin is a protein crucial in angiogenesis, and it is overexpressed in many cancers and downregulated in neurodegenerative diseases, respectively. The protein interaction with actin, through the loop encompassing the 60-68 residues, is an essential step in the cellular cytoskeleton reorganization. This, in turn, influences the cell proliferation and migration processes. In this work, hybrid nanoassemblies of gold nanoparticles with angiogenin fragments containing the 60-68 sequence were prepared and characterized in their interaction with both model membranes of supported lipid bilayers (SLBs) and cellular membranes of cancer (neuroblastoma) and normal (fibroblasts) cell lines. The comparison between physisorption and chemisorption mechanisms was performed by the parallel investigation of the 60-68 sequence and the peptide analogous containing an extra cysteine residue. Moreover, steric hindrance and charge effects were considered with a third analogous peptide sequence, conjugated with a fluorescent carboxyfluorescein (Fam) moiety. The hybrid nanobiointerface was characterized by means of ultraviolet-visible, atomic force microscopy and circular dichroism, to scrutinize plasmonic changes, nanoparticles coverage and conformational features, respectively. Lateral diffusion measurements on SLBs "perturbed" by the interaction with the gold nanoparticles-peptides point to a stronger membrane interaction in comparison with the uncoated nanoparticles. Cell viability and proliferation assays indicate a slight nanotoxicity in neuroblastoma cells and a proliferative activity in fibroblasts. The actin staining confirms different levels of interaction between the hybrid assemblies and the cell membranes.

Gold nanoparticles (AuNPs) have unique anti-angiogenic properties and have been applied in a variety of cancers studies. 'Naked' gold nanoparticles directly bound to angiogenic factors have been demonstrated to inhibit growth factor-mediated signalling in vitro and vascular endothelial growth factor-induced angiogenesis in vivo. Angiogenin (Ang) is a protein physiologically constituent the human plasma but also a pathological marker of different cancer types. Herein, the peptide Ang(60-68), encompassing the putative cellular binding site of the protein Ang, has been synthesized and characterized in the interaction with spherical AuNPs of 12 nm of diameter. The Mg mimicking activity of the peptide was assessed in terms of cell cytoskeleton rearrangements visualized by staining of actin, which is an important target of the whole protein. The hybrid assemblies obtained by physical adsorption of the peptide molecules at the surface of the gold nanoparticles were scrutinised by UV visible spectroscopy, to characterize with titration experiments the changes in the plasmonic properties of AuNPs as well as the peptide spectral features. The latter were obtained by using the fluorescent analogous peptide, Fam-Ang(59-68), incorporating the carboxyfluorescein (Fam) moiety, through an amidic bond of the N-terminal residue. The hydrodynamic size of the peptide-Au systems were determined by dynamic light scattering (DLS) analyses. Proof-of work experiments with human neuroblastoma cells line demonstrated the non-toxicity of the Ang-mimicking peptide functionalised gold nanoparticles. Moreover, laser scanning confocal microscopy (LSM) experiments showed the localization of the peptide-nanoparticles at the cell membrane and their sub-cellular distribution. These data reveal a promising new platform for imaging and therapeutic activities in cancer therapy. (C) 2016 Elsevier B.V. All rights reserved.

Angiogenin (Ang) is a potent angiogenic factor, strongly overexpressed in patients affected by different types of cancers. The specific Ang cellular receptors have not been identified, but it is known that Ang–actin interaction induces changes both in the cell cytoskeleton and in the extracellular matrix. Most in vitro studies use the recombinant form (r-Ang) instead of the form that is normally present in vivo (“wild-type”, wt-Ang). The first residue of r-Ang is a methionine, with a free amino group, whereas wt-Ang has a glutamic acid, whose amino group spontaneously cyclizes in the pyro-glutamate form. The Ang biological activity is influenced by copper ions. To elucidate the role of such a free amino group on the protein–copper binding, we scrutinized the copper(II) complexes with the peptide fragments Ang(1–17) and AcAng(1–17), which encompass the sequence 1–17 of angiogenin (QDNSRYTHFLTQHYDAK-NH2), with free amino and acetylated N-terminus, respectively. Potentiometric, ultraviolet-visible (UV-vis), nuclear magnetic resonance (NMR) and circular dichroism (CD) studies demonstrate that the two peptides show a different metal coordination environment. Confocal microscopy imaging of neuroblastoma cells with the actin staining supports the spectroscopic results, with the finding of different responses in the cytoskeleton organization upon the interaction, in the presence or not of copper ions, with the free amino and the acetylated N-terminus peptides.