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. 2018 Jul 17;57(28):4083-4092.
doi: 10.1021/acs.biochem.8b00457. Epub 2018 Jun 26.

The Structure of an As(III) S-Adenosylmethionine Methyltransferase With 3-Coordinately Bound As(III) Depicts the First Step in Catalysis

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The Structure of an As(III) S-Adenosylmethionine Methyltransferase With 3-Coordinately Bound As(III) Depicts the First Step in Catalysis

Charles Packianathan et al. Biochemistry. .
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Abstract

Arsenic is a ubiquitous environmental toxic substance and a Class 1 human carcinogen. Arsenic methylation by the enzyme As(III) S-adenosylmethionine (SAM) methyltransferase (ArsM in microbes or AS3MT in animals) detoxifies As(III) in microbes but transforms it into more toxic and potentially more carcinogenic methylated species in humans. We previously proposed a reaction pathway for ArsM/AS3MT that involves initial 3-coordinate binding of As(III). To date, reported structures have had only 2-coordinately bound trivalent arsenicals. Here we report a crystal structure of CmArsM from Cyanidioschyzon sp.5508 in which As(III) is 3-coordinately bound to three conserved cysteine residues with a molecule of the product S-adenosyl-l-homocysteine bound in the SAM binding site. We propose that this structure represents the first step in the catalytic cycle. In a previously reported SAM-bound structure, a disulfide bond is formed between two conserved cysteine residues. Comparison of these two structures indicates that there is a conformational change in the N-terminal domain of CmArsM that moves a loop to allow formation of the 3-coordinate As(III) binding site. We propose that this conformational change is an initial step in the As(III) SAM methyltransferase catalytic cycle.

Figures

Figure 1.
Figure 1.
Structure of CmArsM with bound As(III) and SAH. Cartoon diagram (colored salmon) representation of As(III)/SAH-bound CmArsM (PDB entry 6CX6). The overall structure consists of an N-terminal domain, an As(III) binding domain, and a C-terminal domain. The inset shows a close-up of the active site showing the four conserved cysteine residues represented by balls and sticks and colored green (carbon), blue(nitrogen), red (oxygen), and yellow (sulfur). The purple sphere is the arsenic atom, and the SAH in the SAM binding site is represented by balls and sticks and colored magenta (carbon), blue (nitrogen), red (oxygen), and yellow (sulfur). As(III) is bound among conserved residues Cys44, Cys174, and Cys224.
Figure 2.
Figure 2.
Detailed analysis of the As binding site. The As atom is located among conserved residues Cys44, Cys174, and Cys224 in both (A) chain A and (B) chain B of the As(III)/SAH-bound CmArsM structure. The atoms are represented by balls and sticks and colored as in Figure 1, and the distances between atoms are indicated.
Figure 3.
Figure 3.
Distances between the As atom and the sulfur atoms of SAH or SAM in the SAM binding site. (A) The As atom in the binding site consisting of Cys44, Cys174, and Cys224 is positioned near SAH in the SAM binding site of chain A. The distance between the sulfur atom of SAH and the As atom is 4.9 Å. (B) SAM was placed in the binding site by superimposition of the SAM-bound structure with chain A of the As(III)/ SAH-bound structure and substitution of SAM for SAH. At a distance of 2.9 Å, the S-methyl group is poised for electron transfer from SAM to As(III).
Figure 4.
Figure 4.
Movement of conserved cysteine thiols during substrate and/or product binding. (A) Superimposition of As(III)/SAH-bound CmArsM (gray) (PDB entry 6CX6) with unliganded CmArsM (blue) (PDB entry 4FS8) shows that the loop containing Cys72 moves 5.3 Å toward the As binding site when As(III) and SAH are bound. (B) Superimposition of As(III)/SAH-bound CmArsM (gray) with PhAs(III)-bound CmArsM (salmon) (PDB entry 4KW7) shows that the loop moves 5.4 Å in the direction of the As binding site relative to the site with bound PhAs(III). (C) Superimposition of As(III)/SAH-bound CmArsM (gray) with SAM-bound CmArsM (cyan) (PDB entry 4FR0) shows that the loop is 0.9 Å closer to the As binding site when SAH and As(III) are bound compared to when SAM is bound. Conserved cysteine residues, SAM, and SAH are represented as balls and sticks. Cα−Cα distances are indicated.
Figure 5.
Figure 5.
Conformational changes in the ternary structure of CmArsM of the N-terminal domain upon SAM or SAH binding. (A) Unliganded CmArsM (PDB entry 4FS8) and (B) PhAs(III)-bound CmArsM (PDB entry 4KW7) structures have helix H3 between the loop containing Tyr70. (C) SAM-bound CmArsM (PDB entry 4RF0) and (D) As(III)/SAH-bound CmArsM (PDB entry 6CX6) structures lack helix H3. In its place, the residues are part of a large loop that is moved in the direction of the arsenic binding site relative to structures without SAM or SAH. In addition, the position of Tyr70 is oriented away from the SAM binding site, allowing SAM or SAH to access a solvent channel.
Figure 6.
Figure 6.
Superimposition of the N-terminal domains of CmArsM structures. (A) The N-terminal domain of As(III)/SAH-bound CmArsM (gray) and unliganded CmArsM (blue) shows a 6.3 Å movement toward the arsenical binding and a 3.0 Å shift in the loop between helices α1 and α2. The N-terminal domain of PhAs(III)-bound CmArsM (salmon) showed a similar movement relative to the N-terminal domain of the unliganded structure. (B) Superimposition of the N-terminal domain of As(III)/SAH-bound CmArsM (gray) and SAM-bound CmArsM (cyan) shows a 0.5 Å shift in the loop between helices α1 and α2. (C) The N-terminal domains of unliganded (blue) and PhAs(III)-bound (salmon) CmArsM structures are superimposable. Similarly, the N-terminal domains of As(III)/SAH-bound CmArsM (gray) and SAM-bound CmArsM (cyan) structures superimpose well with each other. The conserved cysteines are labeled and colored yellow in the loop of all the structures.
Figure 7.
Figure 7.
Movement of the As binding site during binding of arsenicals. (A) The position of the arsenic atom was analyzed by superposition of the structure of As(III)/SAH-bound CmArsM (PDB entry 6CX6) with that of As(III)-bound CmArsM (PDB entry 4FSD). The arsenic atom in As(III)/SAH bound CmArsM is liganded to Cys44, Cys174, and Cys224 (dashed lines). In the As(III)-bound structure, the arsenic atom is liganded to only Cys174 and Cys224, with a chlorine atom as the third ligand (green sphere). The arsenic atom is 1.1 Å closer to the SAM binding site in the As(III)/SAH-bound structure than in the As(III)-bound structure. (B) Superimposition of the As(III)/SAH-bound CmArsM structure with the MAs(III)-bound structure shows that the arsenic atom in MAs(III) is 1.6 Å distant from the SAM binding site and the methyl group of MAs(III) is facing the solvent channel of the SAM binding site. (C) Similarly, the arsenic atom in the PhAs(III)-bound structure is displaced 1.1 Å relative to its position in the As(III)/SAH-bound structure.
Figure 8.
Figure 8.
Initial steps in the methylation pathway. The first four steps of the pathway leading to methylation of As(III) to MAs(III). In step 1, As(III) is bound to Cys44, Cys174, and Cys224. In step 2, As(III) is oxidatively methylated to form a hypothetical pentavelent intermediate. In step 3, the transient pentavelent intermediate is reduced to enzyme-bound MAs(III) bound to Cys174 and Cys224 with electrons from Cys72, which forms a disulfide bond with Cys44. In step 4, the disulfide bond is reduced by thioredoxin. The bottom panels show structures related to the first four steps: (1) As(III)/SAH-bound CmArsM (PDB entry 6CX6), showing As(III) bound to Cys44, Cys174, and Cys224; (2) SAM-bound CmArsM (PDB entry 4FR0), showing the orientation of the S-methyl group toward the arsenic binding site; (3) formation of the Cys44−Cys72 disulfide bond (PDB entry 4KW7); and (4) the reduced disulfide bond, regenerating Cys44 and Cys72, in MAs(III)-bound CmArsM (PDB entry 5JWN).

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