Escherichia coli BL21(DE3) was used for heterologous protein production as outlined before. All genes were expressed from vector pET-28a(+) (Merck) under control of the T7 promoter, resulting in the addition of an Nterminal hexahistidine (His6)-tag to the heterologously produced proteins. HheC and HheG mutagenesis Amino acid positions for mutagenesis were selected based on the respective motif 1 sequences of HheC (T-X4-F-X-G) and HheG (T-X4-Y-X-G). For each conserved amino acid position within motif 1, variants carrying all 19 possible amino acid exchanges were generated using site-directed mutagenesis (see Table for a list of used primers). In case of positions T13, Y18 and G20 of HheG as well as T7 and G14 of HheC, a Golden Gate mutagenesis protocol was used (see supplementary Table for details regarding the composition of the PCR reaction). The PCR protocol for plasmid amplification while introducing the mutation consisted of an initial denaturation (98 °C, 30 s), 30 cycles of denaturation (98 °C, 10 s), annealing (Tm-5 °C, 30 s) and elongation (72 °C, 30 s kb -1 ), followed by a final elongation step (72 °C, 120 s). After successful generation of mutated linear plasmids, one-pot restriction and ligation was performed using 1x cut smart® buffer (NEB), 1x T4-ligase buffer, 2 U BsaI, 400 U T4-ligase and 150 ng PCR product in 20 µL, and was incubated for 2 h at 30 °C, followed by heat inactivation of the reaction for 20 min at 65 °C. Resulting plasmids were transformed into Escherichia coli BL21(DE3) cells via heat shock method. Correct insertion of desired mutations was confirmed by sequencing. For position F12 of HheC, a MEGAWHOP mutagenesis protocol was used. Respective MEGA-primers were generated using QuikChange® mutagenic reverse primers in combination with a T7 forward primer (Table ). Reaction conditions (see Table ) as well as the PCR protocol were the same as for Golden Gate mutagenesis except for the used annealing condition, which was set to 57 °C for 30 s. For the subsequent MEGAWHOP mutagenesis, 300 ng of purified MEGA-primer, 30 ng pET28a(+)-hheC and PfuUltra II Hotstart PCR Mastermix (Agilent Technologies, Santa-Clara, CA, United States) were applied. The PCR protocol consisted of an initial denaturation step (98 °C, 30 s), 30 cycles of denaturation (98 °C, 10 s), annealing (55 °C, 30 s) and elongation (68 °C, 2 min kb -1 ), followed by a final elongation step (72 °C, 120 s).