title: Capturing Unknown Substrates via in Situ Formation of Tightly Bound Bisubstrate Adducts: S-Adenosyl-vinthionine as a Functional Probe for AdoMet-Dependent Methyltransferases.
abstract: Identifying an enzyme's substrates is essential to understand its function, yet it remains challenging. A fundamental impediment is the transient interactions between an enzyme and its substrates. In contrast, tight binding is often observed for multisubstrate-adduct inhibitors due to synergistic interactions. Extending this venerable concept to enzyme-catalyzed in situ adduct formation, unknown substrates were affinity-captured by an S-adenosyl-methionine (AdoMet, SAM)-dependent methyltransferase (MTase). Specifically, the electrophilic methyl sulfonium (alkyl donor) in AdoMet is replaced with a vinyl sulfonium (Michael acceptor) in S-adenosyl-vinthionine (AdoVin). Via an addition reaction, AdoVin and the nucleophilic substrate form a covalent bisubstrate-adduct tightly complexed with thiopurine MTase (2.1.1.67). As such, an unknown substrate was readily identified from crude cell lysates. Moreover, this approach is applicable to other systems, even if the enzyme is unknown.
authors.forename: Wanlu
authors.initials: W
authors.lastname: Qu
authors.forename: Kalli C
authors.initials: KC
authors.lastname: Catcott
authors.affiliation: School of Pharmaceutical Science and Technology, Tianjin University , Tianjin 300072, China.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Shanshan
authors.initials: S
authors.lastname: Liu
authors.forename: Jason J
authors.initials: JJ
authors.lastname: Guo
authors.affiliation: School of Pharmaceutical Science, Wenzhou Medical University , Wenzhou 325035, China.
authors.forename: Jisheng
authors.initials: J
authors.lastname: Ma
authors.forename: Michael
authors.initials: M
authors.lastname: Pablo
authors.forename: James
authors.initials: J
authors.lastname: Glick
authors.forename: Yuan
authors.initials: Y
authors.lastname: Xiu
authors.forename: Nathaniel
authors.initials: N
authors.lastname: Kenton
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Ma
authors.forename: Richard I
authors.initials: RI
authors.lastname: Duclos
authors.forename: Zhaohui Sunny
authors.initials: ZS
authors.lastname: Zhou
mesh_terms: D002851:Chromatography, High Pressure Liquid; D057930:Click Chemistry; D005001:Ethionine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry* / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000737:chemistry* / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
chemical_list: C020543:vinthionine; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C022745:thiopurine methyltransferase; D005001:Ethionine