A single-domain expansin-like protein from Gloeophyllum trabeum able to cleave xylan

Expansin-related proteins (ERPs) are a broad group of plant cell wall-loosening proteins and are considered non-catalytic as, to date, no cell wall-derived products have been observed as a result of catalysis, despite the presence of a domain that resembles the catalytic domains of GH45 endoglucanases. Here, we report catalytic activity for a single-domain ERP, GtEXPN_133317, from the brown-rot fungus Gloeophyllum trabeum, which is highly expressed in the early phase of spruce colonization. We demonstrate enzyme-dependent formation of xylan-derived products, such as glucuronylated xylo-oligosaccharides, using high-performance anion exchange chromatography with pulsed amperometric detection. Structure-based multiple sequence alignment of ERPs with GH45 endoglucanases showed that, next to a single conserved aspartate (Asp87 in GtEXPN_133317) present in all ERPs and GH45s, fungal ERPs contain a second conserved acidic residue (Asp25 in GtEXPN_133317). Mutation of these two conserved amino acids, Asp87 and Asp25, led to a nearly complete loss of xylanolytic activity. While these findings do not exclude the possibility of a non-catalytic plant cell wall-loosening mechanism, they show that ERPs likely have other modes of action besides what the current paradigm states. Significance statementProtein-mediated plant cell wall-loosening is a natural process that enables plant growth and improves cell wall accessibility for microorganisms. Expansin-related proteins (ERPs) are key to this process, but their mechanism is not fully understood. Traditionally, ERPs are seen as non-catalytic, disrupting non-covalent bonds holding the cellulose network together. We have investigated an expansin-like protein from a fungal saprotroph and demonstrate its catalytic activity by showing product formation from glucuronoxylan and identifying key residues associated with this activity. This activity, although so far only shown for one protein, challenges the current paradigm that ERPs are non-catalytic, shedding new light on plant cell wall architecture and dynamics as well as on the potential roles of ERPs in plant-pathogen interactions.