Fig. 2: Biosynthetic pathway for E-64 and identifying standalone amide-forming enzymes.
From: Enzymatic combinatorial synthesis of E-64 and related cysteine protease inhibitors
a, BGCs of 1 from A. flavus and A. oryzae and other homologous BGCs. The percentage amino acid sequence identity to each corresponding Cp1 enzyme is shown. b, LC–QTOF analysis of metabolites produced by different gene combinations from cp1 and cp2 clusters in the heterologous host A. nidulans is shown in (i)–(v). Selected ion chromatography traces presented on the same scale are shown and the colors of the traces match the indicated mass and compounds. The y axis represents ion counts. *Not isolated. c, Cp1A catalyzed the epoxidation on fumaric acid to t-ES. Assays were carried out at 30 °C for 3 h in 100 μl of 50 mM sodium phosphate buffer with 0.2 mM FeSO4, 2 mM αKG, 2 mM ascorbate, 1 mM fumaric acid and 10 μM Cp1A or MfaA. The products were derivatized with 3-NPH to increase MS sensitivity. Selected ion monitoring of 3-NPH-t-ES ([M + H]+ = 403) is shown. The y axis represents ion counts and the chromatograms are presented on the same scale. d, Enzyme assays with Cp1B and Cp1D. Reactions were performed at 30 °C for 16 h in 100 μl of 50 mM sodium phosphate buffer (pH 8.0). Reaction components for each reaction were as follows: (i) 25 μM Cp1B, 5 mM (±)-t-ES, 2.5 mM l-Ile, 10 mM ATP and 10 mM MgCl2; (ii) 10 μM Cp1D, 2 mM 14, 2.5 mM putrescine, 10 mM ATP and 10 mM MgCl2; (iii) 25 μM Cp1B, 25 μM Cp1D, 5 mM (±)-t-ES, 2.5 mM l-Ile, 5 mM putrescine, 10 mM ATP and 10 mM MgCl2. Traces represent selected ion monitoring of 4 ([M + H]+ = 316) and 14 ([M + H]+ = 246). The y axis represents ion counts and the chromatograms are presented on the same scale. e, Biosynthetic pathway of 1 and the related compounds from the cp1 pathway.
