title: Structure-function roles of four cysteine residues in the human arsenic (+3 oxidation state) methyltransferase (hAS3MT) by site-directed mutagenesis.
abstract: Cysteine (Cys) residues are often crucial to the function and structure of proteins. Cys157 and Cys207 in recombinant mouse arsenic (+3 oxidation state) methyltransferase (AS3MT) are shown to be related to enzyme activity and considered to be the catalytic sites. The roles of some conserved Cys residues in the N-terminal region of the rat AS3MT also have been examined. However, little is known about the roles of the Cys residues in the middle region. The metabolism of inorganic arsenic in human is different from rat and mouse in some aspects though the AS3MT has a high degree of similarity in these species. In order to determine whether the Cys156 and Cys206 (corresponding to the catalytic sites, Cys157 and Cys207 in the mouse AS3MT) in the hAS3MT act as the catalytic sites and to study the roles of the Cys residues (Cys226 and Cys250) near the catalytic center in the middle region, we designed and prepared four mutants (C156S, C206S, C226S, and C250S) in which one Cys residue replaced by serine by PCR-based site-directed mutagenesis. The native form and cysteine/serine mutants were assayed for enzyme activity, free thiols, and the secondary structures by circular dichroism and Fourier transform infrared. Our data show that, besides C156S and C206S, C250S is another potential important site. C226S seems to have the same action as the wild-type hAS3MT with the consistent K(M) and V(max) values. Meanwhile, selenium can also inhibit the methylation of inorganic arsenic by C226S. All the mutants except C226S are calculated to have dramatic changes in the secondary structures. Cys250 might form an intramolecular disulfide bond with another Cys residue. These findings demonstrate that Cys residues at positions 156, 206, and 250 play important roles in the enzymatic function and structure of the hAS3MT.
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Hankou Road 22, Nanjing 210093, PR China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Song
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Geng
authors.forename: Jingshu
authors.initials: J
authors.lastname: Zhu
authors.forename: Chengying
authors.initials: C
authors.lastname: Li
authors.forename: Xin
authors.initials: X
authors.lastname: Hu
authors.forename: Ningsheng
authors.initials: N
authors.lastname: Bian
authors.forename: Xinrong
authors.initials: X
authors.lastname: Zhang
authors.forename: Zhilin
authors.initials: Z
authors.lastname: Wang
mesh_terms: D001151:Arsenic / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000633:toxicity; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012643:Selenium / Q000494:pharmacology; D012694:Serine / Q000235:genetics / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship
chemical_list: D012694:Serine; D008780:Methyltransferases; C505756:AS3MT protein, human; D012643:Selenium; D003545:Cysteine; D001151:Arsenic