title: Identification of the third binding site of arsenic in human arsenic (III) methyltransferase.
abstract: Arsenic (III) methyltransferase (AS3MT) catalyzes the process of arsenic methylation. Each arsenite (iAs(3+)) binds to three cysteine residues, methylarsenite (MMA(3+)) binds to two, and dimethylarsenite (DMA(3+)) binds to one. However, only two As-binding sites (Cys156 and Cys206) have been confirmed on human AS3MT (hAS3MT). The third As-binding site is still undefined. Residue Cys72 in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) may be the third As-binding site. The corresponding residue in hAS3MT is Cys61. Functions of Cys32, Cys61, and Cys85 in hAS3MT are unclear though Cys32, Cys61, and Cys85 in rat AS3MT have no effect on the enzyme activity. This is why the functions of Cys32, Cys61, and Cys85 in hAS3MT merit investigation. Here, three mutants were designed, C32S, C61S, and C85S. Their catalytic activities and conformations were determined, and the catalytic capacities of C156S and C206S were studied. Unlike C85S, mutants C32S and C61S were completely inactive in the methylation of iAs(3+) and active in the methylation of MMA(3+). The catalytic activity of C85S was also less pronounced than that of WT-hAS3MT. All these findings suggest that Cys32 and Cys61 markedly influence the catalytic activity of hAS3MT. Cys32 and Cys61 are necessary to the first step of methylation but not to the second. Cys156 and Cys206 are required for both the first and second steps of methylation. The S(C32) is located far from arsenic in the WT-hAS3MT-SAM-As model. The distances between S(C61) and arsenic in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are 7.5 Å and 4.1 Å, respectively. This indicates that SAM-binding to hAS3MT shortens the distance between S(C61) and arsenic and promotes As-binding to hAS3MT. This is consistent with the fact that SAM is the first substrate to bind to hAS3MT and iAs is the second. Model of WT-hAS3MT-SAM-As and the experimental results indicate that Cys61 is the third As-binding site.
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 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, 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, PR China.
authors.forename: Jiayin
authors.initials: J
authors.lastname: Chang
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, PR China.
authors.forename: Shuping
authors.initials: S
authors.lastname: Wang
authors.affiliation: School of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, PR China.
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Song
authors.affiliation: Modern Analysis Center of Nanjing University, Nanjing, PR China.
authors.forename: Xin
authors.initials: X
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, PR China.
authors.forename: Zhilin
authors.initials: Z
authors.lastname: Wang
mesh_terms: D000818:Animals; D001151:Arsenic / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics*; D002942:Circular Dichroism; D003545:Cysteine / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006801:Humans; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D051381:Rats; D000461:Rhodophyta / Q000201:enzymology; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D017931:DNA Primers; D008780:Methyltransferases; C505756:AS3MT protein, human; D003545:Cysteine; D001151:Arsenic