The pH and temperature profiles were determined via the halide release assay, as described previously , using 10 mM haloalcohol 18 and 25 µg mL -1 purified HheG-682. For both enzymes, the optimal pH for dehalogenation was obtained in phosphate buffer at pH 7.0 (Figure ). In contrast, HheG-682 displayed its highest relative activity at 35 °C (Figure B), which is 5 K higher compared to the reported highest relative activity of HheG in the dehalogenation of 18. For determination of the oligomeric state of HheG-682 in comparison to HheG, size exclusion chromatography on a Superdex 200 Increase 10/300 GL column (GE Healthcare, Freiburg Germany) was performed using 10 mM Tris•SO4 buffer, pH 7.9, containing 4 mM EDTA and 150 mM NaCl at a flow rate of 0.75 mL min -1 . After equilibration of the column, 200 µg of protein was loaded on the column and eluted again in an elution volume of 25 mL. For molecular weight determination, a protein standard mix (15-600 kDa, Sigma-Aldrich, MO USA) was run over the column under the same elution conditions. According to its crystal structure, HheG is known to exhibit a homotetrameric assembly resulting in an overall molecular mass of approx. 120 kDa (29.8 kDa per monomer). As shown in Finally, the crystal structures of HheG (PDB: 5O30) and HheG-682 (PDB: 7WKQ; released by Wan, N.W.) were compared to investigate potential structural reasons for the enhanced catalytic activity of HheG-682. The overall structures of both enzymes are very similar displaying a typical Rossman foldlike architecture (Figure and). A remarkable difference, however, can be observed in the three dimensional arrangement of alpha helices α6 and α7 in HheG-682 (residues 189-212) and HheG (residues 200-223) (Figure ). As previously reported , residue F203 of HheG, which delimits the enzyme active site of HheG together with residues of the catalytic triad, is found in helix α6. Moreover, HheG-682 is missing the flexible loop (spanning residues 39-47 in HheG) that has been reported for HheG previously. Thus, the shape and appearance of the enzyme active sites of HheG and HheG-682 show clear differences, resulting also in slight shifts of the respective catalytic residues (Figure C and D). Such differences will likely explain the observed higher catalytic activity of HheG-682, but further, more detailed analyses will be necessary.