Functional and structural evaluation of cysteine residues in the human arsenic (+3 oxidation state) methyltransferase (hAS3MT).
Arsenic (+3 oxidation state) methyltransferase (AS3MT) catalyzes the methylation of inorganic arsenic (iAs) and plays important role in the detoxication of this metalloid. There are fourteen cysteine residues in the human AS3MT (hAS3MT), among which twelve are absolutely conserved; Cys334 and Cys360 are unique; Cys368 and Cys369 are identified as a CysCys pair. The roles of several conserved cysteine residues in rat AS3MT and hAS3MT have been reported. Herein, the other conserved cysteine residues (Cys72, Cys271, Cys375) and the unique ones (Cys334, Cys360) were systematically replaced by serine using site-directed mutagenesis to study their functions. The mutants were investigated for enzymatic activity, kinetics, thermal stability and secondary structures. Present results indicate that C72S is completely inactive in methylation of iAs and has distinct changes in the secondary structures; Cys72 might form a critical intramolecular disulfide bond with Cys250; Cys271 and Cys375 do not affect the activity and structure of the hAS3MT. However, the mutations of Cys334 and Cys360 can decrease the enzymatic turnovers and change the conformation of the hAS3MT. The kinetic data show that Cys271, Cys334, Cys360 and Cys375 are not involved in the SAM binding. Additionally, all these cysteine residues except Cys375 affect the thermotropic properties of the hAS3MT.
State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Hankou Road 22, Nanjing 210093, PR China.
Xiaoli
X
Song
Zhirong
Z
Geng
Xiangli
X
Li
Qun
Q
Zhao
Xin
X
Hu
Xinrong
X
Zhang
Zhilin
Z
Wang
D018053:Arsenites / Q000378:metabolism; D055162:Biocatalysis; D003545:Cysteine; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017433:Protein Structure, Secondary; D012436:S-Adenosylmethionine / Q000378:metabolism
D018053:Arsenites; D012436:S-Adenosylmethionine; D008780:Methyltransferases; C505756:AS3MT protein, human; D003545:Cysteine; C015001:arsenite