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The genome of the soil Bacteroidota Chitinophaga pinensis encodes a large number of glycoside hydrolases (GHs) with noteworthy features and potentially novel functions. Several are predicted to be active on polysaccharide components of fungal and oomycete cell walls, such as chitin, β-1,3-glucan and β-1,6-glucan. While several fungal β-1,6-glucanase enzymes are known, relatively few bacterial examples have been characterised to date. We have previously demonstrated that C. pinensis shows strong growth using β-1,6-glucan as the sole carbon source, with the efficient release of oligosaccharides from the polymer. We here characterise the capacity of the C. pinensis secretome to hydrolyse the β-1,6-glucan pustulan and describe three distinct enzymes encoded by its genome, all of which show different levels of β-1,6-glucanase activity and which are classified into different GH families. Our data show that C. pinensis has multiple tools to deconstruct pustulan, allowing the species' broad utility of this substrate, with potential implications for bacterial biocontrol of pathogens via cell wall disruption. Oligosaccharides derived from fungal β-1,6-glucans are valuable in biomedical research and drug synthesis, and these enzymes could be useful tools for releasing such molecules from microbial biomass, an underexploited source of complex carbohydrates., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Unlabelled: Chitin synthases (Chs) are responsible for the synthesis of chitin, a key structural cell wall polysaccharide in many organisms. They are essential for growth in certain oomycete species, some of which are pathogenic to diverse higher organisms. Recently, a microtubule interacting and trafficking (MIT) domain, which is not found in any fungal Chs, has been identified in some oomycete Chs proteins. Based on experimental data relating to the binding specificity of other eukaryotic MIT domains, there was speculation that this domain may be involved in the intracellular trafficking of Chs proteins. However, there is currently no evidence for this or any other function for the MIT domain in these enzymes. To attempt to elucidate their function, MIT domains from two Chs enzymes from the oomycete Saprolegnia monoica were cloned, expressed, and characterized. Both were shown to interact strongly with the plasma membrane component, phosphatidic acid, and to have additional putative interactions with proteins thought to be involved in protein transport and localization. Aiding our understanding of these data, the structure of the first MIT domain from a carbohydrate-active enzyme (MIT1) was solved by NMR, and a model structure of a second MIT domain (MIT2) was built by homology modeling. Our results suggest a potential function for these MIT domains in the intracellular transport and/or regulation of Chs enzymes in the oomycetes., Database: Structural data are available in the Biological Magnetic Resonance Bank (BMRB) database under the accession number 19987 and the PDB database under the accession number 2MPK., (© 2016 Federation of European Biochemical Societies.)