Structure-based binding pocket conservation analysis identifies 9-OH urolithin dehydroxylase candidates across phylogenetically diverse bacteria

Urolithins are polyphenolic compounds produced by gut microbiota from dietary ellagitannins and ellagic acid, with urolithin A being the most bioactive and associated with a range of pharmaceutical activities like anti-aging and anti-inflammatory effects. Despite these benefits, the number of known urolithin A-producing bacteria and their corresponding enzymes is very limited. In this study, we combined conventional sequence-based homology search with structural modeling to identify novel bacteria potentially harboring 9-OH urolithin dehydroxylases, primarily characterized in the Enterocloster genus, that catalyze a key dehydroxylation step essential for urolithin A production. Using three Enterocloster references, a sequence database search coupled with subunit co-occurrence screening was performed. AlphaFold 3 was then employed to model the three-dimensional structures of the candidate multi-subunit complexes. Based on these models, comparative analysis was conducted to screen for candidate enzymes that share closely conserved three-dimensional structural characteristics and conserved core residue architectures within their active-site pockets. The resulting non- Enterocloster candidates, including Aromatoleum sp. and Dehalobacter restrictus, exhibited high predicted functional similarity to the reference enzymes based on conserved binding pocket architectures. This study demonstrates that binding pocket structural signatures help the identification of novel potential urolithin dehydroxylases and previously unrecognized urolithin A–producing bacteria.