Mapping and Probing the Substrate Scope of a Ketoamide-Forming Splicease by Computational Design
-Keto-{beta}-amide warheads are protease-inhibiting pharmacophores in peptidomimetic antiviral drugs and drug leads, but their diversity-oriented synthesis for library-based drug discovery can be challenging. The same moiety is installed enzymatically in gene-encoded peptides by tyramine spliceases from spliceotide natural products. However, their substrate scope remains poorly understood. We mined bacterial spliceotide pathways for multi-precursor spliceases and identified HypX from Hyella patelloides that modified 5 of 7 native precursors in E. coli. Single-point mutagenesis showed promiscuity for HypX and the previously identified PlpX but different substrate scopes, suggesting that splicease mining can provide enzymes for focused libraries of various substrate types. To evaluate multipoint variants without exhaustive screening, we combined AlphaFold3 models with Rosetta calculations to rank millions of variants. Top-scoring peptides were spliced more efficiently than single-point mutants. Structural modeling rationalized position-specific tolerance, including a cofactor constraint. This work establishes a mining- and structure-guided strategy to streamline library-based splicease applications.