As described in ref. , the purpose of FuncLib is to be used to design a small set of stable, efficient, and functionally diverse multipoint active-site mutants that are suitable for lowthroughput experimental testing. Our starting point for the FuncLib design was the crystal structure of the most active Kemp eliminase, GNCA4-WT, characterized in our previous work (kcat/KM of 3047 ± 283 M -1 s -1 at pH 7 for the protein with a His-tag) (PDB ID: 5FQK ). This structure was provided as a starting point to the FuncLib server, which is available at . We selected 11 active site positions to diversify, comprising residues in close proximity to the substrate (Figure ). The resulting sequence space is shown in Table . The diversification was performed using the default FuncLib parameters, and the transition state analog 5(6)-nitrobenzotriazole present in the crystal structure was retained as a proxy for the substrate 5-nitrobenzisoxazole. This yielded 3000 variants, ordered by the Rosetta scoring energy (see the Table and the Supplementary Data).