Mapping and Probing the Substrate Scope of a Ketoamide-Forming Splicease by Computational Design

HypX from Hyella patelloides modified 5 of 7 native precursors in E. coli, and its substrate scope differs from PlpX despite both showing promiscuity.

• Why it matters: Keto-{beta}-amide warheads are valuable protease-inhibiting pharmacophores, but enzymatic installation via tyramine spliceases has an unclear substrate range, limiting library design.
• What they did: The study mined bacterial spliceotide pathways to find HypX, tested single-point mutants, then used AlphaFold3 plus Rosetta to rank millions of multipoint variants for splicing efficiency.
• The result: Top-scoring multipoint peptides were spliced more efficiently than single-point mutants, and modeling explained position-specific tolerance, enabling a mining- and structure-guided approach for streamlined splicease libraries.

AI summary of the abstract · may contain errors · not medical advice · read the paper.