Fig. 2: Characterization of BH_MeHis1.0, BH_MeHis1.8, and selected variants.

A Bar chart showing the mean relative conversion achieved along the BH_MeHis1.8 evolutionary trajectory. Biotransformations were performed using 1 (15 mM), 2 (1.5 mM) and enzyme (1.5 µM) in PBS (pH 6.0) with 3% (v/v) MeCN as cosolvent and analyzed by UPLC following 3 h incubation. Error bars represent the standard deviation of measurements made in triplicate centred around the averaged value. To eliminate errors arising from determination of low conversions, BH_MeHis1.0 was monitored over a longer timeframe and conversions were interpolated using linear regression. B Structure showing the amino acid positions mutated in BH_MeHis1.8 (PDB: 8BP0, https://www.rcsb.org/structure/8BP0). Mutations represented as spheres at the C_α and coloured according to their order of introduction, corresponding to the variants shown in Fig. 2A. MeHis23 is shown as atom-coloured sticks with blue carbon atoms. C Bar chart showing the enantiomeric excess of BH_MeHis1.0 (blue) and BH_MeHis1.8 (red) towards the (R)-enantiomer of MBH product 3. Reactions performed using BH_MeHis1.0 (60 µM) or BH_MeHis1.8 (10 µM) with 1 (15 mM), 2 (1.5 mM), PBS pH 6.0 with 20% (v/v) DMSO as cosolvent and analyzed following 23 h incubation. D Michaelis-Menten plot for the MBH reaction between 1 and 2 catalysed by BH_MeHis1.8 (red), BH_MeHis1.0 (blue) and BH32.14 (grey dashed)¹³. Assays were performed at a fixed concentration of 1 (25 mM) and varying concentrations of 2 (0.1–2 mM). Data points shown are averages of triplicate measurements with error bars representing standard deviation. Representative Michaelis-Menten plots at fixed concentrations of 1 and 2 are shown in Supplementary Fig. 5. Source data are provided as a Source Data file.