Investigation Of The Binding Sites Of S-adenosylmethionine And Arsenic In Human Arsenic (Ⅲ) Methyltransferase During Arsenic Methyltion

Arsenic methylation is the main process by which inorganic arsenic (iAs) is metabolized in the organism. In the presence of reductant, arsenic (Ⅲ) methyltransferase (AS3MT) catalyzes the transfer of methyl groups from S-adenosylmethionine (SAM) to the arsenic (As) atom. We studied functions of the acid residues (Asp76, Asp84, Asp102and Asp150) and the residues ranged5A around SAM in human AS3MT (hAS3MT) by homology modeling and experimental methods (such as site-directed mutagenesis, enzyme kinetics and spectroscopy). Functions of Cys residues in hAS3MT were also further studied based on the previous literature and the third binding site of iAsⅢ in hAS3MT was identified. Mechanism of the methyl transfer process is proposed based on the WT-hAS3MT-SAM-As model and the results obtained from this paper and precious studies. The main works of this dissertation are as follows:(1) Residues Asp76, Asp84, Asp102and Asp150in hAS3MT are located in the SAM-binding motifs I (74-ILDLGSGSG-82), Ⅱ (101-IDMT-104) and Ⅲ (147-ESHDIVVSN-155) or in the vicinity of motif I, which have been predicted by multiple-sequence alignments. In order to study functions of the residues, we prepared eight mutants (D76P, D76N, D84P, D84N, D102P, D102N, D150P and D150N) of hAS3MT. Catalytic acticities of the mutants were determined by high performance liquid chromatography-inductively coupled plasma-mass spectrometry (HPLC-ICP-MS), conformations of the mutant were characterized by circular dichroism (CD) and attenuated total reflection Fourier transform infrared spectrometry (ATR-FTIR), and the models of hAS3MT-SAM were built by modeller9v8utilizing the most updated protein template CmArsM. All the mutants except for D150N are completely inactive. The conformations of all mutants differ with that of the wild type (WT)-hAS3MT. In addition, the Kd values suggest that affinities of the mutants for SAM are weakened. The WT-hAS3MT model shows that As-binding sites Cys156and Cys206are located in5A around SAM. Besides D150N, the conformation and microenvironment of the other mutants around SAM differ with those of the WT. Residues Asp76, Asp84, Asp102and Asp150greatly influence the catalytic activity of hAS3MT via affecting SAM-binding or methyl transfer. We demonstrate that Asp76and Asp84probably form hydrogen bonds with SAM indirectly via water molecular, and Asp102significantly affects the SAM-binding via forming hydrogen bonds with SAM. The length of the hydrogen bonds is2.037A and2.081A, respectively. Residue Asp150also influence the catalytic activity of hAS3MT via affecting the the microenvironment of SAM-binding.(2) The WT-hAS3MT-SAM model showed that the residues ranged5A around SAM were57-RYYG-60,76-DLGSGSGRD-84,101-IDMT-104, Q107,134-GYIE-137,155-NCV-157,160-LV-161and C206. The functions of the residues D76, D84, D102, C156and C206have been studied. To determine the functions of overall residues located in5A around SAM, functions of the remaining residues were evaluated and twenty-three mutants R57A, Y58A, Y59A, G60A, L77A, G78A, S79A, G80A, S81A, G82A, R83A, I101A, M103A, T104A, Q107A, G134A, Y135A, I136A, E137A, N155A, V157A, L160A and V161A were obtained by site-directed mutagenesis. Their catalytic activities and conformations were characterized and models were built. The experimental data herein as well as the earlier literature and the model of WT-hAS3MT-SAM show that the SAM-binding domain in hAS3MT comprises residues57-RYYG-60,76-DLGSGSGRD-84,101-IDMT-104,134-GYIE-137,155-NCV-157and160-LV-161.57-RYYG-60, G78, G80, G82and155-NCV-157interact with the methionine of SAM.101-IDMT-104and135-YIE-137are associated with the nucleotide adenosine of SAM. C156and L160are the common residues located between5A around SAM and5A around As. They are in close contact with the methyl transfer process. G78, G80 and G82belonging to the consensus GXGXG located in a loop connecting the first p-strand and the a-helix in Rossmann fold core. Y59, N155, C156and L160orient S+-CH3during its approach to the lone pair of arsenic, and further activate the methyl transfer process. G78, D102, M103, T104,1136and N155form hydrogen bonds with SAM.(3) There are fourteen cysteine residues (Cys32, Cys61, Cys72, Cys85, Cys156, Cys206, Cys226, Cys250, Cys271, Cys334, Cys360, Cys368, Cys369and Cys375) in hAS3MT, among of which have four fully conserved residues Cys32, Cys61, Cys156and Cys206. Except residues Cys32, Cys61and Cys85, functions of other Cys residues have been studied. Each arsenite (iAsⅢ) binds to three cysteine residues. Residue Cys72in Cyanidioschyzon merolae arsenite S-adenosylmethyltransferase (CmArsM) has been proved to be the third As-binding site. The corresponding residue in hAS3MT is Cys61. However, only two As-binding sites (Cys156and Cys206) have been confirmed in hAS3MT. The third As-binding site is still undefined. This is why the functions of Cys32, Cys61and Cys85in hAS3MT merit investigation. Here, three mutants (C32S, C61S and C85S) were designed. Their catalytic activities and conformations were determined. The catalytic activity of C85S is less pronounced than that of WT-hAS3MT. Mutants C32S and C61S are completely inactive in the methylation of iAsⅢ and active in the methylation of monomethylarsonous (MMAⅢ), C156S and C206S are completely inactive in the methylation of iAsⅢ and MMAⅢ, which suggest that Cys32, Cys61, Cys156and Cys206are necessary to the first step of methylation (iAsⅢ to monomethylarsonate (MMA)), while only two residues Cys156and Cys206are required for the second steps of methylation (MMAⅢ to dimethylarsonate acid (DMA)). The SC32is located far from As in the WT-hAS3MT-SAM-As model, and has no chance to bind As, so we propose that the Cys61is the third binding site of iAsⅢ. The distances between SC61and As in WT-hAS3MT-As and WT-hAS3MT-SAM-As models are7.5A and4.1A, respectively, which indicates that SAM-binding to hAS3MT shortens the distance between SC61and As, and promotes As-binding to hAS3MT. This is consistent with the previous result that SAM is the first reactant to bind to hAS3MT and iAs is the second. Mechanism of the methyl transfer process is proposed based on the WT-hAS3MT-SAM-As model, SAM-binding domain and As-binding sites in hAS3MT.