Methylselenol Production Assay via Coupled Enzyme Activity

Our coupled activity assay combines 2 different enzyme activities to detect methylselenol production, i.e., thioredoxin reductase 1 and KYAT1. Briefly, 100 µL of reaction mixture contained 100 mM potassium phosphate buffer pH 7.4, 1–10 mM of MSC, 100 µM of αKG, 100 µM of KMB, 10 µM of PLP, 0.4 µg mammalian TrxR1, 400 µM of NADPH and 50 ng to 400 ng of pure KYAT1 protein or 20 µg of protein from whole cell lysate. The reaction mixture was pre-incubated for 5 min at 37 °C before adding TrxR1 and NADPH. Continuous measurement of NADPH consumption was recorded at 340 nm for every 30 s using a spectrophotometer (PowerWave HT, BioTek). The assay mixture without TrxR1 was used as blank. Under this condition, the molar extinction coefficient was 6220 M⁻¹ cm⁻¹ [19 (link),20 (link)] for NADPH. T_max represents the time taken to consume all the available NADPH in the assay mixture. To assess the inhibitory effects of β-elimination inhibitors on the enzyme activity, the respective compounds were added at the desired concentration as indicated with 100 ng of KYAT1 enzyme.