Abstract text: Proteins of the expansin superfamily (ESPs), including expansins and expansin-like proteins, are ubiquitous cell wall-loosening proteins in plants, fungi and bacteria, contributing to cell wall growth, restructuring and microbial colonization. However, their mechanism of action remains elusive despite their structural resemblance with GH45 glycoside hydrolases. The current paradigm asserts their non-catalytic nature and attributes this to the lack of observable cell wall-derived products that would be expected from catalysis, and to the apparent absence of a second conserved acidic residue that functions as a catalytic base in GH45s. Here, we show that at least one single-domain expansin-like protein from the brown-rot fungus Gloeophyllum trabeum exhibits catalytic activity against beechwood glucuronoxylan. Structure-based sequence alignment of ESPs and GH45s further revealed that, in addition to the aspartate that is conserved in ESPs and GH45s, ESPs harbour a second conserved acidic residue, the type and location of which vary between ESP subfamilies. Mutation of these two aspartates in GtEXPN_133317 (Asp87 and Asp25) resulted in near-complete loss of xylanolytic activity. These results challenge the current ESP paradigm and suggest the co-existence of catalytic and non-catalytic mechanisms.