Efficient selenocysteine-dependent reduction of toxoflavin by mammalian thioredoxin reductase

Toxoflavin (1,6-dimethylpyrimido[5,4-e][1,2,4]triazine-5,7-dione; xanthothricin) is a well-known natural toxin of the pyrimidinetriazinedione type that redox cycles with oxygen under reducing conditions. In mammalian systems, toxoflavin is an inhibitor of Wnt signaling as well as of SIRT1 and SIRT2 activities, but other molecular targets in mammalian cells have been scarcely studied. Interestingly, in a library of nearly 400,000 compounds (PubChem assay ID 588456), toxoflavin was identified as one out of only 56 potential substrates of the mammalian selenoprotein thioredoxin reductase 1 (TrxR1, TXNRD1). This activity was here examined in further detail.

Kinetic parameters in interactions of toxoflavin with rat or human TrxR isoenzymes were determined and compared with those of juglone (5-Hydroxy-1,4-naphthoquinone; walnut toxin) and 9,10-phenanthrene quinone. Selenocysteine dependence was examined using Sec-to-Cys and Sec-to-Ser substituted variants of recombinant rat TrxR1.

Toxoflavin was confirmed as an efficient substrate for TrxR. Rat and human cytosolic TrxR1 supported NADPH-dependent redox cycling coupled to toxoflavin reduction, accompanied by H₂O₂ production under aerobic conditions. Apparent kinetic parameters for the initial rates of reduction showed that rat TrxR1 displayed higher apparent turnover (k_cat = 1700 ± 330 min^-1) than human TrxR1 (k_cat = 1100 ± 82 min^-1) but also a higher Km (K_m = 24 ± 4.3 μM for human TrxR1 versus K_m = 54 ± 18 μM for rat TrxR1). Human TrxR2 (TXNRD2) was less efficient in reduction of toxoflavin (K_m = 280 ± 110 μM and k_cat = 740 ± 240 min^-1). The activity was absolutely dependent upon selenocysteine (Sec). Toxoflavin was also a subversive substrate indirectly inhibiting reduction of other substrates of TrxR1.

Our results identify toxoflavin as an efficient redox cycling substrate of mammalian TrxR enzymes, in a strict Sec-dependent manner.

Тhe interactions of toxoflavin with mammalian TrxR isoenzymes can help to explain parts of the molecular mechanisms giving rise to the well-known toxicity as well as pro-oxidant properties of this toxin.