We hope this will be useful for the field. A plasmid for expressing the constitutively active HOIL-1 variant is available via @addgene.bsky.social: www.addgene.org/229539/

Thanks to all the members of my lab as well as collaborators and co-authors in the Komander (WEHI), Goddard-Borger (WEHI) and Payne (Uni Sydney) labs.

www.biorxiv.org/content/10.1...

We hope that our protocol will enable us and others to generate various ubiquitinated saccharides as tools to further develop the non-proteinaceous ubiquitin field. A plasmid for bacterial expression of the HOIL-1 variant will be available at @addgene.bsky.social soon.

We utilise HOIL's broad saccharide substrate specificity to develop a simple protocol for the chemoenzymatic generation of preparative amounts of ubiquitinated saccharides. Our approach is vastly simplified by our engineered constitutively active HOIL-1 variant.

We find that HOIL-1 efficiently ubiquitinates hydroxyl groups in Ser and a wide variety of mono- and disaccharides. We show that HOIL-1's His510 residues is critical for this activity and that mutation of His510 to Ala switches HOIL-1 activity from hydroxyl groups to targeting amines.

HOIL-1 is an E3 ubiquitin ligase that can ubiquitinate non-proteinaceous substrates such as glycogen and maltose as first shown in work by @iankelsall.bsky.social. In our work, we systematically compare HOIL-1's in vitro activity against a number of amino acids (Lys, Ser, Thr) and saccharides.