title: The functions of crucial cysteine residues in the arsenite methylation catalyzed by recombinant human arsenic (III) methyltransferase.
abstract: Arsenic (III) methyltransferase (AS3MT) is a cysteine (Cys)-rich enzyme that catalyzes the biomethylation of arsenic. To investigate how these crucial Cys residues promote catalysis, we used matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) to analyze Cys residues in recombinant human arsenic (III) methyltransferase (hAS3MT). We detected two disulfide bonds, Cys250-Cys32 and Cys368-Cys369, in hAS3MT. The Cys250-Cys32 disulfide bond was reduced by glutathione (GSH) or other disulfide bond reductants before the enzymatic methylation of arsenite (iAs3+). In addition to exposing residues around the active sites, cleavage of the Cys250-Cys32 pair modulated the conformation of hAS3MT. This adjustment may stabilize the binding of S-Adenosyl-L-methionine (AdoMet) and favor iAs3+ binding to hAS3MT. Additionally, we observed the intermediate of Cys250-S-adenosylhomocysteine (AdoHcy), suggesting that Cys250 is involved in the transmethylation. In recovery experiments, we confirmed that trivalent arsenicals were substrates for hAS3MT, methylation of arsenic occurred on the enzyme, and an intramolecular disulfide bond might be formed after iAs3+ was methylated to dimethylarsinous acid (DMA3+). In this work, we clarified both the functional roles of GSH and the crucial Cys residues in iAs3+ methylation catalyzed by hAS3MT.
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, PR China.
authors.forename: Shuping
authors.initials: S
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, PR China.
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Geng
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, PR China.
authors.forename: Nan
authors.initials: N
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, PR China.
authors.forename: Xiangli
authors.initials: X
authors.lastname: Li
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, Jiangsu, PR China.
authors.forename: Zhilin
authors.initials: Z
authors.lastname: Wang
mesh_terms: D001151:Arsenic / Q000737:chemistry; D018053:Arsenites / Q000737:chemistry*; D002101:Cacodylic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D006801:Humans; D008715:Methionine / Q000737:chemistry; D008780:Methyltransferases / Q000737:chemistry*; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry*; D012435:S-Adenosylhomocysteine / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018053:Arsenites; D004220:Disulfides; D011994:Recombinant Proteins; C472511:dimethylarsinous acid; D012435:S-Adenosylhomocysteine; D008715:Methionine; D002101:Cacodylic Acid; D008780:Methyltransferases; C505756:AS3MT protein, human; D005978:Glutathione; D003545:Cysteine; C015001:arsenite; D001151:Arsenic