Stefanie Neun, Paul Brear, Eleanor Campbell, Theodora Tryfona, Kamel El Omari, Armin Wagner, Paul Dupree, Marko Hyvönen, Florian Hollfelder
Nature Chemical Biology. Online ahead of print. 07 July 2022 Jul.
DOI: 10.1038/s41589-022-01071-x
Pubmed: 35799064
PDB coordinates:
7QE1 (3D view), 7QE2 (3D view), 7QEF (3D view), 7QEA (3D view), 7QG4 (3D view), 7QEE (3D view)
Abstract
The abundance of recorded protein sequence data stands in contrast to the small number of experimentally verified functional annotation. Here we screened a million-membered metagenomic library at ultrahigh throughput in microfluidic droplets for β-glucuronidase activity. We identified SN243, a genuine β-glucuronidase with little homology to previously studied enzymes of this type, as a glycoside hydrolase 3 family member. This glycoside hydrolase family contains only one recently added β-glucuronidase, showing that a functional metagenomic approach can shed light on assignments that are currently ‘unpredictable’ by bioinformatics. Kinetic analyses of SN243 characterized it as a promiscuous catalyst and structural analysis suggests regions of divergence from homologous glycoside hydrolase 3 members creating a wide-open active site. With a screening throughput of >107 library members per day, picolitre-volume microfluidic droplets enable functional assignments that complement current enzyme database dictionaries and provide bridgeheads for the annotation of unexplored sequence space.