Gencheva Radosveta, Cheng Qing, Arnér Elias S J
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77 Stockholm, Sweden.
Biochim Biophys Acta Gen Subj. 2018 Nov;1862(11):2511-2517. doi: 10.1016/j.bbagen.2018.05.014. Epub 2018 May 25.
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 HO production under aerobic conditions. Apparent kinetic parameters for the initial rates of reduction showed that rat TrxR1 displayed higher apparent turnover (k = 1700 ± 330 min) than human TrxR1 (k = 1100 ± 82 min) but also a higher Km (K = 24 ± 4.3 μM for human TrxR1 versus K = 54 ± 18 μM for rat TrxR1). Human TrxR2 (TXNRD2) was less efficient in reduction of toxoflavin (K = 280 ± 110 μM and k = 740 ± 240 min). 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.
毒黄素（1,6 - 二甲基嘧啶并[5,4 - e][1,2,4]三嗪 - 5,7 - 二酮；黄原黄素）是一种著名的嘧啶三嗪二酮类天然毒素，在还原条件下可与氧气进行氧化还原循环。在哺乳动物系统中，毒黄素是Wnt信号通路以及SIRT1和SIRT2活性的抑制剂，但对哺乳动物细胞中其他分子靶点的研究却很少。有趣的是，在一个近40万种化合物的文库（PubChem分析ID 588456）中，毒黄素被鉴定为哺乳动物硒蛋白硫氧还蛋白还原酶1（TrxR1，TXNRD1）仅有的56种潜在底物之一。在此对该活性进行了更详细的研究。
测定毒黄素与大鼠或人TrxR同工酶相互作用的动力学参数，并与胡桃醌（5 - 羟基 - 1,4 - 萘醌；核桃毒素）和9,10 - 菲醌的动力学参数进行比较。使用重组大鼠TrxR1的硒代半胱氨酸（Sec）到半胱氨酸（Cys）和Sec到丝氨酸（Ser）取代变体研究硒代半胱氨酸依赖性。
毒黄素被确认为TrxR的有效底物。大鼠和人胞质TrxR1支持与毒黄素还原偶联的NADPH依赖性氧化还原循环，在有氧条件下伴有HO产生。还原初始速率的表观动力学参数表明，大鼠TrxR1的表观周转率（k = 1700±330 min）高于人TrxR1（k = 1100±82 min），但Km也更高（人TrxR1的K = 24±4.3 μM，大鼠TrxR1的K = 54±18 μM）。人TrxR2（TXNRD2）还原毒黄素的效率较低（K = 280±110 μM，k = 740±240 min）。该活性绝对依赖于硒代半胱氨酸（Sec）。毒黄素也是一种间接抑制TrxR1其他底物还原的颠覆性底物。
我们的结果表明毒黄素是哺乳动物TrxR酶的一种有效的氧化还原循环底物，且严格依赖于Sec。
毒黄素与哺乳动物TrxR同工酶的相互作用有助于解释该毒素产生众所周知的毒性以及促氧化特性的部分分子机制。
