Protein production in 500 mL scale and purification via FPLC For kinetic analysis of HheG M45F as well as crystallization of HheG M45F-D114C, both variants were produced in 500 mL scale according to the protocol described above, and purified via their N-terminal His-tag using fast protein liquid chromatography (FPLC). Cell disruption was performed as described above but using 30 mL buffer A (50 mM Tris•SO4, 300 mM Na2SO4, 25 mM imidazole, pH 7.9), supplemented with 1 mg mL -1 lysozyme and 1 Pierce Protease Inhibitor Mini Tablet, for cell resuspension. Cell free extracts were loaded with a flow rate of 2 mL min -1 on a 5 mL HisTrap FF column (GE Healthcare, Freiburg, Germany), preequlibrated with lysis buffer, using an ÄktaStart FPLC system (GE Healthcare). Afterwards, the column was washed with 10 CV of buffer A to remove other proteins. His-tagged protein was eluted using a gradient over 60 mL to 100% buffer B (50 mM Tris•SO4, 300 mM Na2SO4, 500 mM imidazole, pH 7.9) while collecting 1 mL fractions. Fractions with highest UV absorbance were pooled and desalted using a HiPrep 26/10 desalting column (GE Healthcare), pre-equilibrated with TE buffer (10 mM Tris•SO4, 4 mM EDTA, pH 7.9, 10% (v/v) glycerol). During desalting, protein was eluted using TE buffer. Desalted protein fractions with highest UV absorbance were concentrated using Vivaspin Turbo 15 centrifugation units (Sartorius, Göttingen, Germany) with 10 kDa molecular weight cut-off. Protein concentrations of resulting purified protein solutions were determined as described above. Both HheG variants were stored at -20 °C until further use.