The Ustilago maydis AA10 LPMO is active on fungal cell wall chitin.

Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and ß-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle.IMPORTANCELytic polysaccharide monooxygenases (LPMOs) have been mainly studied in a biotechnological context for the efficient degradation of recalcitrant polysaccharides. Only recently, alternative roles and paradigms begin to emerge. In this study, we provide evidence that the AA10 LPMO from the phytopathogen Ustilago maydis is active against fungal cell wall chitin. Given that chitin-active LPMOs are commonly found in microbes, it is important to consider fungal cell wall as a potential target for this enigmatic class of enzymes.

On the expansion of biological functions of lytic polysaccharide monooxygenases.

Lytic polysaccharide monooxygenases (LPMOs) constitute an enigmatic class of enzymes, the discovery of which has opened up a new arena of riveting research. LPMOs can oxidatively cleave the glycosidic bonds found in carbohydrate polymers enabling the depolymerisation of recalcitrant biomasses, such as cellulose or chitin. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. In the present review, we propose a historical perspective of LPMO research providing a succinct overview of the major achievements of LPMO research over the past decade. This journey through LPMOs landscape leads us to dive into the emerging biological functions of LPMOs and LPMO-like proteins. We notably highlight roles in fungal and oomycete plant pathogenesis (e.g. potato late blight), but also in mutualistic/commensalism symbiosis (e.g. ectomycorrhizae). We further present the potential importance of LPMOs in other microbial pathogenesis including diseases caused by bacteria (e.g. pneumonia), fungi (e.g. human meningitis), oomycetes and viruses (e.g. entomopox), as well as in (micro)organism development (including several plant pests). Our assessment of the literature leads to the formulation of outstanding questions, promising for the coming years exciting research and discoveries on these moonlighting proteins.

Insights into peculiar fungal LPMO family members holding a short C-terminal sequence reminiscent of phosphate binding motifs.

Lytic polysaccharide monooxygenases (LPMOs) are taxonomically widespread copper-enzymes boosting biopolymers conversion (e.g. cellulose, chitin) in Nature. White-rot Polyporales, which are major fungal wood decayers, may possess up to 60 LPMO-encoding genes belonging to the auxiliary activities family 9 (AA9). Yet, the functional relevance of such multiplicity remains to be uncovered. Previous comparative transcriptomic studies of six Polyporales fungi grown on cellulosic substrates had shown the overexpression of numerous AA9-encoding genes, including some holding a C-terminal domain of unknown function ("X282"). Here, after carrying out structural predictions and phylogenetic analyses, we selected and characterized six AA9-X282s with different C-term modularities and atypical features hitherto unreported. Unexpectedly, after screening a large array of conditions, these AA9-X282s showed only weak binding properties to cellulose, and low to no cellulolytic oxidative activity. Strikingly, proteomic analysis revealed the presence of multiple phosphorylated residues at the surface of these AA9-X282s, including a conserved residue next to the copper site. Further analyses focusing on a 9 residues glycine-rich C-term extension suggested that it could hold phosphate-binding properties. Our results question the involvement of these AA9 proteins in the degradation of plant cell wall and open new avenues as to the divergence of function of some AA9 members.