Biochemical, kinetic, and spectroscopic characterization of Ruegeria pomeroyi DddW - A mononuclear iron-dependent DMSP lyase

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW

C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells

Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC) in control and hyperglycemic conditions.HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86)Rb(+)) uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM). DNA binding activity was determined by electrical mobility shift assay (EMSA). Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase α(1)-subunit protein expression, accompanied with increase in (86)Rb(+) uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1)-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6), concomitant with Na,K-ATPase α(1)-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(1)-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C-peptide-mediated signaling. Importantly, only physiological concentrations of C-peptide elicit this effect

The role of cholesterol metabolism and various steroid abnormalities in autism spectrum disorders : A hypothesis paper

© 2017 The Authors Autism Research published by Wiley Periodicals, Inc. on behalf of International Society for Autism Research.Peer reviewedPublisher PD

View Points: Partnering for Rangeland Health on Tribal Lands

The Rangelands archives are made available by the Society for Range Management and the University of Arizona Libraries. Contact [email protected] for further information.Migrated from OJS platform March 202

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

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

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 (GX₅HXHX_3,4EX₆G) and 2 (GX₅PXGX₂HX₃N), 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

Stoichiometry of Fe(II) binding to DddW.

<p>2 μM apo-DddW (under tight-binding conditions) was titrated with increasing concentrations of Fe(NH₄)₂(SO₄)₂ 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

Dependence of initial velocity (V_i) 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(NH₄)₂(SO₄)₂ and 300μM MnCl₂. 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_max = 36.50 ± 1.27 μM/s; k_cat = 18.25 s^-1; <i>K</i>_m = 8.68 ± 0.73 mM; <i>k</i>_cat/<i>K</i>_m = 2.10 x 10³ M^-1s^-1; With Mn(II): V_max = 34.66 ± 1.64 μM/s; k_cat = 17.33 s^-1; <i>K</i>_m = 4.50 ± 0.75 mM; <i>k</i>_cat/<i>K</i>_m = 3.85x10³ M^-1s^-1.</p