Fig. 3: Stereoselective (2S,3S)-t-ES-l-amino acid synthesis by Cp1B and Cp2B.

a, Crystal structure of Cp1B in a complex with adenosine and MES. Domains A (residues 5–200 and 444–502), B (residues 201–323) and C (residues 365–443) are shown as deep salmon, wheat and cyan, respectively. The helical connection (residues 324–364) between domains B and C is shown in gray. The P-loop and A-loop are highlighted in purple and blue, respectively. Adenosine is colored in light green and MES is colored in cyan. mF_o − DF_c polder omit maps for adenosine and MES are shown in gray mesh and contoured at 3.0σ. b, Enlarged view of the substrate-binding site. Conserved residues with hGSH synthetases are labeled in red, which are proposed to be involved in phosphorylation of substrate carboxylate. Residues surrounding MES that are possibly involved in substrate binding are shown in blue. c, Amino acid nucleophile (proteinogenic amino acids and a1–a32) scope for Cp1B and Cp2B. The heat map shows percentage yields in analytical-scale reactions catalyzed by Cp1B and Cp2B, estimated from the standard curves generated at λ = 204 nm of the purified compounds. The analytical-scale reactions were performed in 100 μl of 50 mM sodium phosphate buffer (pH 8.0) at 30 °C for 16 h. Each reaction contained 25 μM enzyme, 5 mM (±)-t-ES, 2.5 mM amino acid, 10 mM MgCl₂ and 10 mM ATP. Isolated percentage yields from the preparative-scale reactions catalyzed using either Cp1B or Cp2B are shown under the structures. Preparative-scale reactions were performed in 20 ml of 50 mM sodium phosphate buffer (pH 8.0) at 30 °C for 16 h. Each reaction contained 2.5 μM Cp1B or Cp2B, 5 mM (±)-t-ES, 2.5 mM amino acid, 10 mM MgCl₂ and 10 mM ATP. Notes on percentage yields: (i) isolated percentage yields with Cp1B; (ii) isolated percentage yields with Cp2B; (iii) analytical percentage yield estimated from the standard curves generated at λ = 204 nm of purified 14 is shown because t-ES-Met was not isolated from the preparative-scale reaction in this study.