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.2010 Sep 7;49(35):7683-93.
doi: 10.1021/bi100458d.

Structural insight into methyl-coenzyme M reductase chemistry using coenzyme B analogues

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Structural insight into methyl-coenzyme M reductase chemistry using coenzyme B analogues

Peder E Cedervall et al. Biochemistry..

Abstract

Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F(430). Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F(430) nickel in the deepest part of the 30 A long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 A predominantly hydrophobic part of the channel close to F(430), with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 A. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB(5)SH, CoB(6)SH, CoB(8)SH, and CoB(9)SH, respectively). The data presented here reveal that the shorter CoB(5)SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB(6)SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB(6)SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB(8)SH and CoB(9)SH analogues can be accommodated in the active site through exclusion of this water. These analogues react with Ni(III)-methyl, a proposed MCR catalytic intermediate of methanogenesis. The CoB(8)SH thiolate is 2.6 A closer to the nickel than that of CoBSH, but the additional carbon of CoB(9)SH only decreases the nickel thiolate distance a further 0.3 A. Although the analogues do not induce any structural changes in the substrate channel, the thiolates appear to preferentially bind at two distinct positions in the channel, one being the previously observed CoBSH thiolate position and the other being at a hydrophobic annulus of residues that lines the channel proximal to the nickel.

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Figures

Figure 1
Figure 1
The active site and substrate channel in the MCRox1-silent crystal structure (PDB code 1hbn) (9). Coenzyme F430, CoBSH and HSCoM are drawn as stick colored by atom (carbon: dark grey). The nickel is displayed as a green sphere, water as a red sphere. Interactions are drawn as dashed lines, and the corresponding distance is indicted in Angstroms (Å). The path of the substrate channel was defined in the absence of F430, CoBSH, HSCoM and water, with the surface closest to the viewer cut away. The figure was generated using PyMOL (http://www.pymol.org).
Figure 2
Figure 2
Drawing of CoBSH analogues; (A)N-5-mercaptopentanoylthreonine phosphate (CoB5SH); (B)N-6-mercaptohexanoylthreonine phosphate (CoB6SH); (C)N-8-mercaptooctanoylthreonine phosphate (CoB8SH); (D)N-9-mercaptononanoylthreonine phosphate (CoB9SH).
Figure 3
Figure 3
The active sites and substrate channels of the MCR crystal structures.; (A) MCRCoB5SH; (B) MCRCoB6SH; (C) MCRHSCoM; (D) MCRCoB8SH; (E) MCRCoB9SH. 2Fo-Fc electron density map around the CoBXSH analogues, waters in the CoBSH binding part of the channel and the acetate ion (contoured at 1σ) is shown as a blue mesh. The protein is drawn as cartoon. CoBXSH and acetate are drawn as stick and colored by atom (carbon: CoB5SH orange; CoB6SH pale yellow; CoB8SH light blue; CoB9SH magenta; acetate white). Coenzyme F430 and HSCoM are drawn as stick colored by atom (carbon: F430 dark grey; HSCoM medium grey). The nickel is displayed as a green sphere, and waters as red spheres. The figure was generated using PyMOL (http://www.pymol.org/).
Figure 4
Figure 4
Overlay of CoBSH (from PDB code 1hbn) and the different CoBSH analogues. CoBXSH are drawn as stick with the thiol represented by a sphere, and colored CoB5SH orange; CoB6SH pale yellow; CoBSH light green; CoB8SH light blue; CoB9SH magenta. The protein is drawn as cartoon. Coenzyme F430 and HSCoM are drawn as stick colored by atom (carbon: F430 dark grey; HSCoM medium grey). The nickel is displayed as a green sphere. The figure was generated using PyMOL (http://www.pymol.org/).
Figure 5
Figure 5
Hydrogen bonding diagram for the water structure modeled in MCRHSCoM. The water molecules are named as in Figure 3C (W1–W9); WA, WB and WC are water molecules that are present in all structures (i.e. in concert with the substrate CoBSH and the CoBSH analogues). Interactions between surrounding residues and the water molecules are drawn as dashed lines, and the corresponding distance is indicated in Angstroms (Å).
Figure 6
Figure 6
Stereo image of the annulus of aromatic amino acids distal of coenzyme F430. The protein is drawn as cartoon with the side-chains of the aromatic residues drawn as white stick. CoBSH, (from PDB code 1hbn (9)), CoB8SH and CoB9SH are drawn as stick with the thiols represented by spheres, and colored CoBSH light green; CoB8SH light blue; CoB9SH magenta. Coenzyme F430 and HSCoM are drawn as stick colored by atom (carbon: F430 dark grey; HSCoM medium grey). The nickel is displayed as a green sphere. The figure was generated using PyMOL (http://www.pymol.org/).
Scheme 1
Scheme 1
Reaction catalyzed by methyl-coenzyme M reductase
Scheme 2
Scheme 2
Two of the proposed catalytic mechanisms for methyl-coenzyme M reductase; (A) mechanism I; (B) mechanism II.
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References

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