8 research outputs found
The pH dependence of DddW lyase activity.
<p>The optimal pH was determined by comparing the initial velocities (V<sub>i</sub>) of reactions containing 2 μM apo-DddW, 2 μM Fe(II), and 10 mM DMSP in varying buffer solutions. The buffers used are as follows: 50 mM MES 20 mM NaCl (pH 5.5, 6.0, 6.5), 50 mM HEPES 20 mM NaCl (pH 7.0, 7.5, 8.0), 50 mM Tris-HCl 20 mM NaCl (pH 8.5, 9.0).</p
Stoichiometry of Fe(II) binding to DddW.
<p>2 μM apo-DddW (under tight-binding conditions) was titrated with increasing concentrations of Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub> and the fluorescence intensity was monitored. The titration data were analyzed by nonlinear curve fitting using Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127288#pone.0127288.e003" target="_blank">3</a>) to produce the solid line. Upon data fitting, the stoichiometric ratio of Fe(II) to DddW monomer was determined to be 1:1.</p
Proposed mechanisms for the mononuclear iron dependent DMSP lyase, DddW.
<p>DddW binds to Fe(II) cofactor to which the substrate can coordinate in either monodentate or bidentate modes. (A) His81 can act as a nucleophile to remove a hydrogen atom from the α-carbon of DMSP to form acrylate. (B) A hypothetical water molecule can be activated by His81, which then acts as a nucleophile in initiating catalysis. (C) Tyr89 located near the active site can initiate the elimination reaction cleaving DMSP.</p
Cupin motifs and metal binding residues of DddW.
<p>(A) Sequence alignment of cupin regions of selected DddW, DddQ and DddL proteins using sequences deposited at NCBI and CLUSTAL 2.1 for the alignment. The two conserved cupin motifs 1 (GX<sub>5</sub>HXHX<sub>3,4</sub>EX<sub>6</sub>G) and 2 (GX<sub>5</sub>PXGX<sub>2</sub>HX<sub>3</sub>N), containing residues that bind metal ions and are catalytically important are highlighted in green. Tyr residues playing catalytic role in <i>Ruegeria lacuscaerulensis</i> DddQ are marked cyan and other conserved residues in the cupin motifs are colored yellow. The sequences are from: W1 = DddW, <i>Ruegeria pomeroyi</i> DSS-3 (SPO0453); W2 = DddW, <i>Roseobacter sp</i>. MED193, (MED193_09710); Q1 = DddQ, <i>Ruegeria pomeroyi</i> DSS-3 (SPO1596); Q2 = DddQ, <i>Ruegeria lacuscaerulensis</i> (ITI-1157); L1 = DddL, <i>Sulfitobacter sp</i>. EE-36 (EE36_11918); L2 = DddL, <i>Rhodobacter sphaeroides</i> 2.4.1 (RSP_1433); L3 = DddL, <i>Roseibacterium elongatum</i> DSM 19469 (roselon_02436); L4 = DddL, <i>Caenispirillum salinarum</i> (C882_2645). (B) Homology model of <i>Ruegeria pomeroyi</i> DddW (grey) (generated using Phyre 2 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127288#pone.0127288.ref052" target="_blank">52</a>]) superimposed on the Zn(II)-bound structure of <i>Ruegeria lacuscaerulensis</i> DddQ (cyan) (PDB 4LA2). The homology model of DddW shows the catalytic residues H81, H83, E87, and H121. Most of these residues of DddW (H83, E87, and H121) superimpose well on the zinc-coordinating DddQ residues (H125, E129, and H163). While Tyr usually is not involved in metal ion binding in cupin proteins, the DddQ structure shows a Zn-coordinated Tyr residue (Tyr131) and this Tyr superimposes on Tyr89 of DddW. The side chain residues are shown in ball and stick with oxygens in red, nitrogens in blue, zinc in slate, and carbons are similar to protein backbone.</p
Spectral properties of Fe(II)-bound DddW.
<p>(A) UV-visible spectra of the reaction of as-isolated DddW in the presence of Fe(II) and Cu(II). All spectra with Fe(II) had an enzyme concentration of 370 μM. Trace in black, apo-DddW, green, apo-DddW in presence of 370 μM Fe(II) red, apo-DddW+Fe(II) after bubbling with NO gas. The absorption maximum is at 340 nm with a shoulder at 430 nm. Inset: Spectrum of 1 mM apo-DddW in the presence of Cu(II). The spectral feature at 550 nm is due to a charge transfer transition of DddW with Cu(II). (B) EPR spectra of: (top) 18 μM apo-DddW with Fe(II); (bottom) Fe(II)-DddW in the presence of 25 mM DMSP. The spectra were collected at microwave frequency, 9.43 GHz; receiver gain, 2 x 104; modulation frequency, 100 kHz; temperature, 4 K; microwave power, 200 microwatts; 83.89 s sweep time, and 16 scans.</p
Dependence of initial velocity (V<sub>i</sub>) of DddW catalyzed lyase reaction on DMSP concentrations in the presence of Fe(II) and Mn(II).
<p>Apo-DddW (2 μM) was mixed with an equimolar amount of Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub> and 300μM MnCl<sub>2</sub>. To this reaction mixture, varying concentrations (0.5–35 mM) of DMSP was added. The reactions were monitored at 205 nm. The data were fit to the Michaelis-Menten equation. The kinetic parameters are as follows. With Fe(II): V<sub>max</sub> = 36.50 ± 1.27 μM/s; k<sub>cat</sub> = 18.25 s<sup>-1</sup>; <i>K</i><sub>m</sub> = 8.68 ± 0.73 mM; <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 2.10 x 10<sup>3</sup> M<sup>-1</sup>s<sup>-1</sup>; With Mn(II): V<sub>max</sub> = 34.66 ± 1.64 μM/s; k<sub>cat</sub> = 17.33 s<sup>-1</sup>; <i>K</i><sub>m</sub> = 4.50 ± 0.75 mM; <i>k</i><sub>cat</sub>/<i>K</i><sub>m</sub> = 3.85x10<sup>3</sup> M<sup>-1</sup>s<sup>-1</sup>.</p
Metal binding affinities (K<sub>d</sub>) of DddW.
<p>Titration of apo-DddW with increasing concentrations of the metal ions Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub> (added anaerobically), FeCl<sub>3</sub>, CoCl<sub>2</sub>, MnCl<sub>2</sub>, CuCl<sub>2</sub> or NiCl<sub>2</sub> was done. The binding was monitored by saturation of the fluorescence intensities. The concentration of enzyme used is as follows: (A) 0.5 μM apo-DddW, (B)-(F) 2 μM apo-DddW. The K<sub>d</sub> values were: (A) Fe(II), 4.7 ± 0.0 nM; (B) Fe(III), 89.3 ± 4.3 μM; (C) Mn(II), 32.7 ± 5.0 μM; (D) Co(II), 2.5 ± 0.4 μM; (E) Ni(II), 1.0 ± 0.1 μM; (F) Cu(II), 1.9 ± 0.2 μM.</p
DMSP lyase activity of DddW variants compared to wild-type enzyme.
<p>Activity assays were performed anaerobically using 2 μM DddW, 2 μM Fe(NH<sub>4</sub>)<sub>2</sub>(SO<sub>4</sub>)<sub>2</sub> and 10 mM DMSP. The reactions were incubated for 15 mins before quenching.</p