Crystal structure of the SPOC domain of <i>A</i>. <i>thaliana</i> FPA.

<p><b>(A)</b>. Schematic drawing of the structure of FPA SPOC domain, colored from blue at the N terminus to red at the C terminus. The view is from the side of the β-barrel. The disordered segment (residues 460–465) is indicated with the dotted line. (<b>B</b>). Structure of the FPA SPOC domain, viewed from the end of the β-barrel, after 90° rotation around the horizontal axis from panel A. All structure figures were produced with PyMOL (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>).</p

Impact of individual FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA.

<p>RNA gel blot analysis of WT <i>A</i>. <i>thaliana</i> accession Columbia (Col-0) plants <i>fpa-8</i> and <i>fpa-8</i> mutants expressing either <i>FPA</i>::<i>FPA R477A</i> <b>(A)</b>, or <i>FPA</i>::<i>FPA Y515A</i> <b>(B)</b> using poly(A)+ purified mRNAs. A probe corresponding to the 5’UTR region of <i>FPA</i> mRNA was used to detect <i>FPA</i> specific mRNAs. RNA size (kb) marker (Ambion). <i>TUBULIN</i> was detected as an internal control. Proximally and distally polyadenylated <i>FPA</i> transcripts are marked with arrows. The ratio of distal:proximal polyadenylated forms is given under each lane. <b>(C,D)</b> Impact of individual FPA SPOC domain mutations on <i>FLC</i> transcript levels. qRT-PCR analysis was performed with total RNA purified from Col-0, <i>fpa-8</i>, <i>35S</i>::<i>FPA</i>:<i>YFP</i> and <i>FPA</i>::<i>FPA R477A</i> <b>(C)</b>, <i>FPA</i>::<i>FPA Y515A</i> <b>(D)</b> plants. Transcript levels were normalized to the control <i>UBC</i>. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</p

A conserved surface patch of FPA SPOC domain.

<p><b>(A)</b>. Two views of the molecular surface of FPA SPOC domain colored based on sequence conservation among plant FPA homologs. Purple: most conserved; cyan: least conserved. <b>(B)</b>. Residues in the conserved surface patch of FPA SPOC domain. The side chains of the residues are shown in stick models, colored orange in the first sub-patch and green in the second. <b>(C)</b>. Molecular surface of FPA SPOC domain colored based on electrostatic potential. Blue: positively charged; red: negatively charged.</p

Structural homologs of the FPA SPOC domain.

<p><b>(A)</b>. Overlay of the structures of the FPA SPOC domain (cyan) and the SHARP SPOC domain (gray). The bound position of a doubly-phosphorylated peptide from SMRT is shown in magenta. <b>(B)</b>. Overlay of the structures of the FPA SPOC domain (cyan) and the Ku70 β-barrel domain (gray). Ku80 contains a homologous domain (green), which forms a hetero-dimer with that in Ku70. The two domains, and inserted segments on them, mediate the binding of dsDNA (orange). The red rectangle highlights the region of contact between the two β-barrel domains. <b>(C)</b>. Overlay of the structures of the FPA SPOC domain (cyan) and the homologous domain in Chp1 (gray). The binding partner of Chp1, Tas3, is shown in green. The red rectangle indicates the region equivalent to the binding site of the SMART phosphopeptide in SHARP SPOC domain, where a loop of Tas3 is also located. (<b>D</b>). Overlay of the structures of the FPA SPOC domain (cyan) and the Med25 ACID (gray).</p

Impact of double FPA SPOC domain mutations on alternative polyadenylation of FPA pre-mRNA and <i>FLC</i> expression.

<p><b>(A)</b> RNA gel blot analysis of WT <i>A</i>. <i>thaliana</i> accession Columbia (Col-0) plants <i>fpa-8</i> and <i>fpa-8</i> mutants expressing <i>FPA</i>::<i>FPA R477A;Y515A</i> using poly(A)+ purified mRNAs. Black arrows indicate the proximally and distally polyadenylated <i>FPA</i> mRNAs. A probe corresponding to the 5’UTR region of <i>FPA</i> mRNA was used to detect <i>FPA</i> specific mRNAs. RNA size (kb) marker (Ambion). <i>TUBULIN</i> was detected as an internal control. The ratio of distal:proximal polyadenylated forms is given under each lane. <b>(B)</b>. qRT-PCR analysis was performed with total RNA purified from Col-0, <i>fpa-8</i>, and <i>FPA</i>::<i>FPA R477A;Y515A</i> plants. Transcript levels were normalized to the control <i>UBC</i>. Histograms show mean values ±SE for three independent PCR amplifications of three biological replicates.</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

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

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

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

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