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. 2018 Mar;3(3):3104-3112.
doi: 10.1021/acsomega.8b00197. Epub 2018 Mar 14.

Reorientation of the Methyl Group in MAs(III) Is the Rate-Limiting Step in the ArsM As(III) S-Adenosylmethionine Methyltransferase Reaction

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Free PMC article

Reorientation of the Methyl Group in MAs(III) Is the Rate-Limiting Step in the ArsM As(III) S-Adenosylmethionine Methyltransferase Reaction

Charles Packianathan et al. ACS Omega. .
Free PMC article

Abstract

The most common biotransformation of trivalent inorganic arsenic (As(III)) is methylation to mono-, di-, and trimethylated species. Methylation is catalyzed by As(III) S-adenosylmethionine (SAM) methyltransferase (termed ArsM in microbes and AS3MT in animals). Methylarsenite (MAs(III)) is both the product of the first methylation step and the substrate of the second methylation step. When the rate of the overall methylation reaction was determined with As(III) as the substrate, the first methylation step was rapid, whereas the second methylation step was slow. In contrast, when MAs(III) was used as the substrate, the rate of methylation was as fast as the first methylation step when As(III) was used as the substrate. These results indicate that there is a slow conformational change between the first and second methylation steps. The structure of CmArsM from the thermophilic alga Cyanidioschyzon merolae sp. 5508 was determined with bound MAs(III) at 2.27 Å resolution. The methyl group is facing the solvent, as would be expected when MAs(III) is bound as the substrate rather than facing the SAM-binding site, as would be expected for MAs(III) as a product. We propose that the rate-limiting step in arsenic methylation is slow reorientation of the methyl group from the SAM-binding site to the solvent, which is linked to the conformation of the side chain of a conserved residue Tyr70.

Conflict of interest statement

Notes The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Analysis of the first and second steps of CmArsM methylation. Methylation was assayed in a final volume of 1 mL containing 1 mM SAM, 5 mM GSH, 2 μM purified CmArsM, and 10 μM of either As(III) (A) or MAs(III) (B) at 60 °C, as described under Materials and Methods. Samples were withdrawn at the indicated times, and the reaction were terminated by the addition of 10% (v/v) H2O2, final concentration. Arsenicals were speciated by HPLC-ICP-MS. Inset: As(III) methylation at shorter times.
Figure 2
Figure 2
As(III) methylation at 37 °C. Methylation was assayed as described in the legend to Figure 1 except that the temperature was 37 °C.
Figure 3
Figure 3
Methylation activity with either As(III) or MAs(III). Methylation activity was assayed at 37 °C by conversion of SAM to SAH using a EPIgeneous methyltransferase assay kit (Cisbio Bioassays, Bedford, MA), as described under Materials and Methods. The assay was carried out for the indicated times in 384-well microtiter plates in a buffer consisting of 50 mM NaH2PO4, pH 8.0, containing 0.3 M NaCl, 2 mM GSH, 1 μM purified CmArsM and 10 μM of either As(III) or MAs(III). SAH production was estimated from the HTRF.
Figure 4
Figure 4
Structure of CmArsM with bound MAs(III). Cartoon diagram (colored in light orange) representation of MAs(III)-bound CmArsM (PDB ID 5JWN). The overall structure consists of an N-terminal domain, As(III) binding domain, and a C-terminal domain. Inset: the expanded view of the active site shows the conserved cysteine residues represented by ball-and-stick and colored green (carbon), blue (nitrogen), red (oxygen), or yellow (sulfur). The purple sphere is the arsenic atom, and the light blue sphere is a Ca2+ ion found in the SAM-binding site. MAs(III) is bound between conserved residues Cys174 and Cys224.
Figure 5
Figure 5
Tyr70 gates substrate access to the active site. (A) Superposition of wild-type CmArsM structures with bound MAs(III) (light orange) and bound SAM (light gray) indicates that the main chain (Cα–Cα) of Tyr70 differs by 4.1 Å in the two structures. In the SAM-bound structure, Tyr70 is closer to the As(III) binding site than in the MAs(III)-bound structure, suggesting that the hydroxyphenyl side chain of Tyr70 sterically hinders binding of arsenicals when SAM is bound. (B) The surface representation of the MAs(III)-bound structure (light gray) shows that the Tyr70 is in the solvent channel oriented toward the As-binding site and a SAM molecule modeled into the SAM-binding site in ball-and-stick. (C) Surface representation of SAM-bound wild-type CmArsM in the same orientation. In this structure, the side chain of Tyr70 is oriented away from the SAM-binding site, allowing SAM and SAH access to the active site.
Figure 6
Figure 6
Modeling the complex of CmArsM with MAs(III) and SAM. (A) Stereo view of the superimposition of the MAs(III)-bound (light orange) and SAM-bound (light gray) structures, with an rmsd of 1.25 Å. The ternary complex of CmArsM with bound MAs(III) and SAM was modeled by superposition of their individual structures. The distances from the S-methyl group of SAM to the sulfur atom of conserved cysteine residues and to the arsenic atom of MAs(III) are indicated. (B) The PhAs(III)-bound C72A structure was superimposed with the SAM-bound wild-type CmArsM structure, with an rmsd of 1.25 Å. The distances of the S-methyl group of SAM to the sulfur atom of conserved residues Cys174 and Cys224 and to the arsenic atom of PhAs(III) are indicated. The atoms representation and the coloring are the same as that described in Figure 4.
Figure 7
Figure 7
Detailed analysis of the As-binding site. (A) MAs(III) is located between conserved residues Cys174 and Cys224 in the MAs(III)-bound structure. In the pyramidal binding site, the distances between the As atom of MAs(III) and the sulfur thiolates of Cys174 and Cys224 are 2.5 and 2.4 Å, respectively. The thiolate of Cys72 is 7.3 Å away from the arsenic atom of MAs(III). The carbon atom of MAs(III) serves as a third arsenic ligand. Each of the liganding atoms are at an average distance of 3.7 Å from each other. (B) PhAs(III) in the C72A structure is located between Cys174 and Cys224. The distances between As atom of PhAs(III) and the sulfur thiolates of Cys174 and Cys224 are 2.5 and 2.3 Å, respectively. In this pyramidal binding site, the central arsenic atom is coordinated with Cys174 and Cys224 at an average distance of 2.4 Å and the C1 carbon atom of PhAs(III) at an average distance of 3.4 Å. Both the methyl group of MAs(III) and the hydroxyphenyl ring of PhAs(III) are oriented toward the solvent channel. The atoms are represented by ball and stick and colored as in Figure 4.
Figure 8
Figure 8
Reorientation of the As-methyl group is rate-limiting for catalysis. (A) As(III) is positioned between its binding site composed of Cys174 and Cys224 and the SAM S-methyl group. (B) The methyl group is transferred from SAM to As(III), forming a MAs(III)-bound intermediate in which the As-methyl group is oriented toward SAH. (C) Slow reorientation of the As-methyl group toward bulk solvent opens the SAM-binding site, allowing exchange of SAH for SAM. In this conformation, MAs(III) becomes the substrate for the second round of methylation.

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