Biochemical assays of StnR, StnQ1, StnK3

(1). Assays for the StnR activity with l-Trp were performed in a 100 μL solution at 30 °C for 1 h in the presence of 1 mM l-Trp, 1 mM α-ketoglutaric acid (α-KG) and 5 μM StnR in 50 mM 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES, pH 8.0) buffer.

(2). Assays for the StnQ1 activity with indolepyruvate (InPy) as the substrate were performed in a 100 μL solution at 30 °C for 1 h. The assay mixtures contained 50 mM HEPES buffer (pH 8.0), 1 mM InPy, 1 mM S-adenosylmethionine (SAM) and 5 μM StnQ1.

(3). Assays for the StnQ1/R or StnQ1/R/K3-catalyzed coupled reaction with l-Trp as the substrate were performed in a 100 μL solution at 30 °C for 1 h in the presence of 1 mM l-Trp, 1 mM SAM and 5 μM StnQ1, 5 μM StnR, and 5 μM StnK3 in 50 mM HEPES buffer (pH 8.0).

(4). Assays for the StnK3 reaction with (2S,3R)-β-MeTrp as a surrogate were performed in a 100 μL solution at 30 °C for 2 hours in the presence of 1 mM (2S,3R)-β-MeTrp, and 10 μM StnK3 in 50 mM HEPES buffer (pH 8.0).

(5). Assays for the StnR activity with racemic β-methyl indolepyruvate were performed in a 100 μL solution at 30 °C for 1 hour in the presence of 1 mM β-methyl InPy, 1 mM l-Trp, and 20 μM StnR or 2 μM StnR in 50 mM HEPES buffer (pH 8.0).

All of the reactions were quenched by addition of 200 μL methanol to precipitate proteins and solvent was removed by speed-vac. The resulting residues were re-dissolved in 80 μL methanol, and analyzed with HPLC or LC-HRMS in positive mode. The mobile phase was comprised of solvent A (Milli-Q water) and solvent B (acetonitrile). HPLC analyses were performed on an Agilent HPLC Series 1200 and LC-HRMS analyses were performed on an Agilent 1200 series coupled with a 6530 Accurate-Mass Q-TOF MASS Spectrometer using a ZORBAX SB-C18 (Agilent, 5 μm, 150 × 4.6 mm) column under the following conditions: 5% to 40% B (0–15 min); 40–100% B (15–20 min); 100% B (20–25 min); 100% to 5% B (25–26 min); 5% B (26–30 min) at a flow rate of 0.6 mL min⁻¹ (0.5 mL min⁻¹ for LC-MS and LC-HR-MS) and UV detection at 280 nm. When detecting β-methyl indolepyruvate acid, solvent A contained 1‰ formic acid and the flow rate was changed to 0.3 mL min⁻¹.