Amino acid positions were selected based on their position at the crystal interface. Symmetry mates of HheG were generated using PyMOL (version 2.3.3, Schrödinger, New York, NY, United States). This way, amino acids facing each other within a distance of less than 5 Å at the crystal interface were selected by the PRODIGY crystal webserver. Site-directed mutagenesis was performed using the PfuUltra II Hotstart PCR Mastermix (Agilent Technologies, Santa-Clara, CA, United States). Forward and reverse mutagenic primers (Table ) were designed with PrimerX (Carlo Lapid, 2003, ), purchased from Merck and used in concentrations of 0.25 µM each with 100 ng of pET28a(+)-hheG template. Otherwise, the mutagenesis protocol was in agreement with the manufacturer's instructions.