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. Author contributionCD designed the research and coordinated the project. Molecular biology (cloning) was done by AM and CD. AM and BEK expressed and purified the protein. BEK performed fluorescence experiments. AM and AJS recorded EPR spectra. AJS simulated the EPR spectra. AM and BEK ran activity assays on chitin. MF performed mass spectrometry experiments. MF, HR, and DR analyzed mass spectrometry data. CD and BEK crystallized the protein. AR collected diffraction data at the synchrotron. CD and AR solved the crystal structure. Bioinformatic analysis was carried out by AM and CD. All the data were compiled and analyzed by CD, JS, and MR. All authors contributed to the writing and approved the final version of the manuscript.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.

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