Initial design was performed using the FuncLib webserver (), as described in ref. . As our starting point, we selected all amino acids in close contact with the substrate for randomization by FuncLib, comprising of 11 starting positions (V48, D50, I250, R256, L260, V261, L285, V286, V287, W290 and H291, see Table ). The calculations were performed on Chain A of the crystal structure of the GNCA4-W229D/F290W variant (PDB ID: 5FQK, henceforth referred to as GNCA4-WT), with the transition state analog, 6nitrobenzotriazole, retained in the calculation, and the His tag removed. The multiple sequence alignment was performed using the default parameters, and the top twenty ranked designs based on their stability score were retained for further experimental and computational analysis.