title: Substrate profiling of glutathione S-transferase with engineered enzymes and matched glutathione analogues.
abstract: The identification of specific substrates of glutathione S-transferases (GSTs) is important for understanding drug metabolism. A method termed bioorthogonal identification of GST substrates (BIGS) was developed, in which a reduced glutathione (GSH) analogue was developed for recognition by a rationally engineered GST to label the substrates of the corresponding native GST. A K44G-W40A-R41A mutant (GST-KWR) of the mu-class glutathione S-transferases GSTM1 was shown to be active with a clickable GSH analogue (GSH-R1) as the cosubstrate. The GSH-R1 conjugation products can react with an azido-based biotin probe for ready enrichment and MS identification. Proof-of-principle studies were carried to detect the products of GSH-R1 conjugation to 1-chloro-2,4-dinitrobenzene (CDNB) and dopamine quinone. The BIGS technology was then used to identify GSTM1 substrates in the Chinese herbal medicine Ganmaocongji.
authors.affiliation: MOE Key Laboratory of Bioinformatics, School of Life Sciences, Renhuan Building 301, Tsinghua University, 100084 Beijing (China).
authors.forename: Shan
authors.initials: S
authors.lastname: Feng
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Gulishana
authors.initials: G
authors.lastname: Adilijiang
authors.forename: Jieyuan
authors.initials: J
authors.lastname: Liu
authors.forename: Minkui
authors.initials: M
authors.lastname: Luo
authors.forename: Haiteng
authors.initials: H
authors.lastname: Deng
mesh_terms: D057930:Click Chemistry; D004365:Drugs, Chinese Herbal / Q000737:chemistry / Q000378:metabolism; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013058:Mass Spectrometry; D008958:Models, Molecular; D040081:Protein Array Analysis; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D004365:Drugs, Chinese Herbal; D005982:Glutathione Transferase
keywords: click chemistry; glutathione S-transferases; mass spectrometry; protein engineering; substrate profiling