title: Synthesis of l-cysteine derivatives containing stable sulfur isotopes and application of this synthesis to reactive sulfur metabolome.
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 synthesized34S-labeled L-cysteine from O-acetyl-L-serine and34S-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 with15N-labeled L-serine, acetyl-CoA, and34S-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 prepared34S-labeled N-acetyl-L-cysteine (NAC) by incubating34S-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 using34S-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.
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Ono
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai 980-8577, Japan.
authors.forename: Minkyung
authors.initials: M
authors.lastname: Jung
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Tianli
authors.initials: T
authors.lastname: Zhang
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Hiroyasu
authors.initials: H
authors.lastname: Tsutsuki
authors.affiliation: Life Sciences and Applied Markets Group, Agilent Technologies, Tokyo 192-8510, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sezaki
authors.affiliation: Department of Biological Science, Graduate School of Science, Osaka Prefecture University, Osaka 599-8531, Japan.
authors.forename: Hideshi
authors.initials: H
authors.lastname: Ihara
authors.affiliation: Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan; Precursory Research for Embryonic Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan.
authors.forename: Fan-Yan
authors.initials: FY
authors.lastname: Wei
authors.affiliation: Department of Molecular Physiology, Faculty of Life Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
authors.forename: Kazuhito
authors.initials: K
authors.lastname: Tomizawa
authors.affiliation: Department of Environmental Health Sciences and Molecular Toxicology, Tohoku University Graduate School of Medicine, Sendai 980-8577, Japan.
authors.forename: Takaaki
authors.initials: T
authors.lastname: Akaike
authors.affiliation: Department of Microbiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan. Electronic address: sawat@kumamoto-u.ac.jp.
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Sawa
mesh_terms: D000111:Acetylcysteine / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D003547:Cysteine Synthase / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007553:Isotope Labeling; D055442:Metabolome; D010084:Oxidation-Reduction; D012486:Salmonella typhimurium / Q000201:enzymology / Q000378:metabolism*; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013455:Sulfur / Q000378:metabolism; D013460:Sulfur Isotopes
chemical_list: D013438:Sulfhydryl Compounds; D013460:Sulfur Isotopes; D013455:Sulfur; D003547:Cysteine Synthase; D003545:Cysteine; D000111:Acetylcysteine
keywords: Glutathione persulfide; Isotope labeling; L-cysteine persulfide; Mass spectrometry; N-acetyl-L-cysteine; Reactive sulfur species; Sulfur metabolome