Synthesis of L-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome
Synthesis of L-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome
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DOI：
10.1016/j.freeradbiomed.2017.02.023
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发表时间：
2017-05-01
影响因子：
7.4
通讯作者：
Sawa, Tomohiro
中科院分区：
文献类型：
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作者：
Ono, Katsuhiko;Jung, Minkyung;Sawa, Tomohiro
半胱氨酸过硫化物是一种L-半胱氨酸衍生物，含有一个额外的硫原子与半胱氨酸硫醇结合，它是一种活性硫物种，调节细胞内氧化还原动态平衡。本文介绍了一种快速、高效地合成含同位素硫原子的L-半胱氨酸衍生物的方法，并将该方法应用于活性硫代谢物。我们使用细菌半胱氨酸合成法将同位素硫原子引入到L-半胱氨酸的巯基部分。我们从革兰氏阴性菌鼠伤寒沙门氏菌LT2中克隆了三种半胱氨酸合成酶CysE、CysK和CysM，并制备了它们的重组酶。我们以0-乙酰基-L丝氨酸和34S标记的硫化钠为底物合成了34S标记的L半胱氨酸，用于CysK或CysM反应。在CysE和CysK存在下，通过与15N标记的L丝氨酸、乙酰辅酶A和34S标记的硫化钠进行酶反应，实现了L半胱氨酸在硫(34S)和氮(15N)原子上的同位素标记。该酶体系可用于合成一系列L-半胱氨酸衍生物，包括L-半胱氨酸、L-过硫半胱氨酸、S-磺基-L-半胱氨酸、半胱氨酸磺酸盐和L-硒半胱氨酸。我们还将34S标记的L半胱氨酸与乙酰辅酶A在试管中孵育，制备了34S标记的N-乙酰-L半胱氨酸(NAC)。单溴莫比胺衍生化后的低分子硫醇的串联质谱学鉴定表明，在培养的哺乳动物细胞如HeLa细胞和J774.1细胞中存在内源性NAC。此外，我们还利用~(34)S标记的NAC，成功地证明了NAC在细胞内通过中间产物L-半胱氨酸的形成代谢转化为谷胱甘肽及其过硫化物。因此，使用同位素硫标记和质谱学相结合的方法可能有助于更好地了解活性硫代谢组和氧化还原生物学。
Cysteine persulfide is an L-cysteine derivative having one additional sulfur atom bound to a cysteinyl thiol group, and it serves as a reactive sulfur species that regulates redox homeostasis in cells. Here, we describe a rapid and efficient method of synthesis of L-cysteine derivatives containing isotopic sulfur atoms and application of this method to u reactive sulfur metabolome. We used bacterial cysteine syntheses to incorporate isotopic sulfur atoms into the sulfhydryl moiety of L-cysteine. We cloned three cysteine synthases CysE, CysK, and CysM from the Gram-negative bacterium Salmonella enterica serovar Typhimurium LT2, and we generated their recombinant enzymes. We synthesized 34S-labeled L-cysteine from 0-acetyl-L-serine and 34S-labeled sodium sulfide as substrates for the CysK or CysM reactions. Isotopic labeling of L-cysteine at both sulfur (34S) and nitrogen (15N) atoms was also achieved by performing enzyme reactions with 15N-labeled L-serine, acetyl-CoA, and 34S-labeled sodium sulfide in the presence of CysE and CysK. The present enzyme systems can be applied to syntheses of a series of L-cysteine derivatives including L-cystine, L-cystine persulfide, S-sulfo-L-cysteine, Lcysteine sulfonate, and L-selenocystine. We also prepared 34S-labeled N-acetyl-L-cysteine (NAC) by incubating 34S-labeled L-cysteine with acetyl coenzyme A in test tubes. Tandem mass spectrometric identification of low molecular-weight thiols after monobromobimane derivatization revealed the endogenous occurrence of NAC in the cultured mammalian cells such as HeLa cells and J774.1 cells. Furthermore, we successfully demonstrated, by using 34S-labeled NAC, metabolic conversion of NAC to glutathione and its persulfide, via intermediate formation of L-cysteine, in the cells. The approach using isotopic sulfur labeling combined with mass spectrometry may thus contribute to greater understanding of reactive sulfur metabolome and redox biology.