The active site of DmdD.

<p>(<b>A</b>). Omit F_o–F_c electron density map at 1.8 Å resolution for MTA-CoA in binding mode A, contoured at 3σ. (<b>B</b>). Omit F_o–F_c electron density map for MTA-CoA in binding mode B, contoured at 2.5σ. (<b>C</b>). Overlay of the two DmdD active sites in the crystal of the E121A mutant in complex with MTA-CoA (in stereo). For binding mode A, molecule 1 of DmdD is shown in cyan, molecule 3 in yellow, and MTA-CoA in green. The MTA-CoA molecule in binding mode B and the corresponding binding residues in DmdD are shown in gray. The red arrow indicates the shift in the position of MTA-CoA in binding mode B relative to binding mode A. The blue arrow indicates conformational differences for the β9-α5 loop (include Glu141) between the two DmdD molecules. Close neighbors of the sulfur atom in binding mode A are indicated with the dashed lines (yellow). (<b>D</b>). Solvent accessible surface of the active site region of DmdD, corresponding to binding mode A of MTA-CoA (in green). (<b>E</b>). Overlay of binding modes A (in green) and B (in light cyan) of MTA-CoA and binding mode A of MMPA-CoA (in gray).</p

Summary of kinetic parameters.

1<p>Values shown are the average and standard deviation from triplicate experiments. Acetyl-CoA, acryloyl-CoA, butyryl-CoA, isobutyryl-CoA, malonyl-CoA and pentanoyl-CoA were also tried as substrates for CoA ester hydrolysis, but no activity was observed. In addition, acryloyl-CoA was not hydrated.</p>2<p>N.A. – No activity was detected. The limits for detection were 1.4×10⁻⁴ s⁻¹ and 4.8×10⁻⁵ s⁻¹ for CoA ester hydrolysis and MeSH release, respectively.</p

Sequence conservation of DmdD.

<p>(<b>A</b>). The MeSH and DMS catabolic pathways of DMSP. The enzymes in the MeSH parthway are indicated. The chemical structures of acryloyl-CoA, crotonyl-CoA and pentanoyl-CoA are shown in the inset. (<b>B</b>). Sequence alignment of <i>Ruegeria pomeroyi</i> DmdD, <i>Myxococcus xanthus</i> DmdD, <i>Pseudomonas aeruginosa</i> DmdD, and rat liver enoyl-CoA hydratase (ECH). The secondary structure elements in the structure of <i>R. pomeroyi</i> DmdD are labeled. The two catalytic Glu residues are indicated with the blue triangles. The residue numbers refer to <i>R. pomeroyi</i> DmdD.</p

Structure of the DmdD monomer.

<p>(<b>A</b>). Overlay of the structures of the two DmdD (E121A mutant) molecules in complex with MTA-CoA in the asymmetric unit of the crystal. One molecule is colored in cyan, and the other in gray. The MTA-CoA molecules are shown in stick models. The red boxes highlight regions of structural differences between the two molecules. (<b>B</b>). Omit F_o–F_c electron density map at 1.8 Å resolution, contoured at 3σ, for residues 121 and 148–150 in the structure of the E121A mutant of DmdD (in cyan) in complex with MTA-CoA (in green). The structure of these residues in wild-type DmdD is shown for comparison (in gray). The flip of the Gly149–Gly150 peptide bond is likely linked to the E121A mutation. All structure figures were produced with PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p

Reactions of DmdD with crotonyl-CoA and 3-hydroxybutyryl-CoA.

<p>Chromatography the DmdD reaction products following incubation with 160 µM crotonyl-CoA for 0, 10, and 50 min (<b>A</b>) or 3-hydroxybutyryl-CoA (3-HB-CoA) for 0 and 30 min (<b>B</b>). AU is absorbance units. (<b>C</b>). Reaction time course for the hydration/hydrolysis of crotonyl-CoA by DmdD. Crotonyl-CoA (▵) is consumed at an initial rate of 90 µmol min⁻¹ mg⁻¹. The formation of 3-hydroxybutyryl-CoA (□) and HS-CoA (○) occurs at initial rates of 72 µmol min⁻¹ mg⁻¹ and 12 µmol min⁻¹ mg⁻¹, respectfully. After 2 min the rate of consumption of crotonyl-CoA proceeds at 0.94 µmol min⁻¹ mg⁻¹. Consumption of 3-hydroxybutyryl-CoA occurs at 2.5 µmol min⁻¹ mg⁻¹, while formation of free CoA proceeds at 3.3 µmol min⁻¹ mg⁻¹.</p

A proposed catalytic mechanism for the hydration and hydrolysis of MTA-CoA by DmdD.

<p>The products of the reaction are shown in red. For the hydrolysis of the anhydride in the anhydride mechanism, the hydroxyl group can attack either of the carbonyl carbons, as indicated by the two arrows.</p

Structure of the DmdD hexamer.

<p>(<b>A</b>). Structure of the DmdD E121A mutant hexamer in complex with MTA-CoA, viewed down the three-fold symmetry axis (black triangle). The three molecules of the trimer are given different colors, and the MTA-CoA molecules are shown in stick models (in green). The N-terminal domain (NTD), C-terminal domain (CTD), and C-terminal loop (CTL) are labeled. (<b>B</b>). Structure of the DmdD hexamer, viewed from the side, down a two-fold symmetry axis (black oval). The six molecules of the hexamer are labeled. (<b>C</b>). Overlay of the structures of the DmdD E121A mutant monomer (in cyan) in complex with MTA-CoA (in green) and rat liver ECH monomer in complex with AcAc-CoA (in gray) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063870#pone.0063870-Engel1" target="_blank">[15]</a>. A large difference in the conformation of the C-terminal loop (CTL) of the two structures is indicated in red. (<b>D</b>). Structure of the rat liver ECH hexamer in comple with AcAc-CoA, viewed down the three-fold symmetry axis (black triangle). The CoA binding region is more open to the solvent compared to that in DmdD (panel A).</p

Summary of crystallographic information.

1<p>The numbers in parentheses are for the highest resolution shell.</p