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

Abstract

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.

Decoding the Ambiguous Electron Paramagnetic Resonance Signals in the Lytic Polysaccharide Monooxygenase from Photorhabdus luminescens

“… Structure of the Pl AA10 LPMO enzyme (PDB code: 6T5Z) 15 and of its active site, with special focus on the copper center and its first coordination sphere. Bond distances shown in Å.…”

Identification of Polyvalent Vaccine Candidates From Extracellular Secretory Proteins in Vibrio alginolyticus

“…LPMOs act synergistically with glycoside hydrolases such as cellulases and chitinases by oxidatively cleaving several glycosidic bonds at the surface of their crystalline substrate(s). In addition to their role in biomass degradation, some bacterial LPMOs act as virulence factors in several human and insect pathogens ( 34 ). AT730_22220 is carotenoid 1,2-hydratase (CrtC), which catalyzes the selective addition of water to an isolated carbon-carbon double bond ( 35 ).…”

On the expansion of biological functions of lytic polysaccharide monooxygenases

“…Literature analysis of the occurrence of lytic polysaccharide monooxygenases (LPMOs) found in various pathosystems. (a, b) Number of research articles reporting an LPMO (or LPMO‐like) shown to be relevant by either transcriptomic, proteomic and/or enzyme characterisation in different emitters (Kawada et al ., 2008; Chaudhuri et al ., 2010; Valente et al ., 2011; Stauder et al ., 2012; Wong et al ., 2012; Yakovlev et al ., 2012; Garcia‐Gonzalez et al ., 2014; Loose et al ., 2014; Chiu et al ., 2015; Hamre et al ., 2015; Huang et al ., 2015; Paspaliari et al ., 2015; Zhang et al ., 2015; Mekasha et al ., 2016; Shukla et al ., 2016; Liu et al ., 2017, 2018, 2020; Hegnar et al ., 2019; Yadav et al ., 2019; Garcia‐Santamarina et al ., 2020; Labourel et al ., 2020; Li et al ., 2020; Munzone et al ., 2020; Askarian et al ., 2021; Polonio et al ., 2021; Sabbadin et al ., 2021a,b; Yue et al ., 2021), and displayed as (a) a function of the year of publication per emitter and (b) an emitter–target map, in which the circle size indicates the number of articles and the colour indicates the protein family. Note that the ‘preferred target’ shows the potential target of the emitter, and not necessarily the target of the LPMO, which in several cases remains to be fully demonstrated (see main text).…”