To study the impact of motif 1 mutations on the activity, selectivity and stability of HHDHs, each of the three conserved residues of this motif (T-X4-F/Y-X-G) in HheC and HheG was individually replaced by the corresponding other 19 proteinogenic amino acids using either a Golden Gate-based or MEGAWHOP mutagenesis strategy. This resulted in a total of 112 defined single mutants, of which all could be generated successfully, except for mutants F12M and F12R of HheC. Subsequent heterologous production of all 110 mutants in E. coli BL21(DE3) in 15 mL scale and partial purification via N-terminal His-tag using gravity flow revealed that only few variants per position, usually carrying exchanges to structurally similar amino acids, still yielded observable amounts of soluble HHDH (Figure and Figure ). Hence, this result suggests a possible direct impact of motif 1 residues on protein folding and stability. As an exception, position F12 of HheC permitted more diverse mutations as almost all variants could be obtained as soluble proteins (Figure and Figure ).