Synthesis of L-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome

Katsuhiko Ono, Minkyung Jung, Tianli Zhang, Hiroyasu Tsutsuki, Hiroshi Sezaki, Hideshi Ihara, Fan Yan Wei, Kazuhito Tomizawa, Takaaki Akaike, Tomohiro Sawa

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

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 a 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 O-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, L-cysteine 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.

Original languageEnglish
Pages (from-to)69-79
Number of pages11
JournalFree Radical Biology and Medicine
Volume106
DOIs
Publication statusPublished - May 1 2017
Externally publishedYes

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Keywords

  • Glutathione persulfide
  • Isotope labeling
  • L-cysteine persulfide
  • Mass spectrometry
  • N-acetyl-L-cysteine
  • Reactive sulfur species
  • Sulfur metabolome

ASJC Scopus subject areas

  • Biochemistry
  • Physiology (medical)

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