Psc 718

Reduction of RidA by thioredoxin was followed spectrometrically at the absorption maximum of NADPH using a JASCO V-650 spectrophotometer equipped with the temperature-controlled cell holder PSC-718. An amount of 50 μM RidA were mixed with 5 μM TrxA (Sigma-Aldrich, St Louis, USA) and 100 μM NADPH (Sigma-Aldrich, St. Louis, USA) in 50 mM Tris-buffer, pH 7.5. After 30 s of stirring, 80 nM TrxB were added. Consumption of NADPH was measured at 340 nm for 2 h.
Reduction by glutathione was carried out by incubating RidA_HOCl with a 1-fold or 10-fold molar excess of glutathione (GSH or GSSG) for 30 min at 37 °C before the measurement of chaperone holdase activity.

Analysis of α-ketobutyrate generation was performed as described previously21 (link)23 (link). Briefly, RidA was treated with HOCl and/or DTT as described in the main body of the manuscript. After the removal of reagents, 1.8 μM RidA were mixed with 0.9 μM IlvA in 1 × reaction buffer (50 mM MES, 50 mM HEPES, 50 mM TAPS pH 7.5). The reaction was started by the addition of 10 mM threonine. α-Ketobutyrate generation was followed at 230 nm in a JASCO V-650 spectrophotometer equipped with the temperature-controlled cell holder PSC-718 (JASCO, Tokyo, Japan) using a 3.5 ml QS-macro-cuvette (10 mm) with a magnetic stir bar at 20 °C.

All chemicals were purchased from Sigma-Aldrich (St. Louis, United States). For activity assays, a Jasco V-650 spectrophotometer, equipped with a PSC-718 temperature-controlled sample holder (Jasco Corporation, Tokyo, Japan) was used.