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

Structural and Biochemical Insights into Dimethylsulfoniopropionate Cleavage by Cofactor-bound DddK from the Prolific Marine Bacterium Pelagibacter

Enormous amounts of the organic osmolyte dimethylsulfoniopropionate (DMSP) are produced in marine environments where bacterial DMSP lyases cleave it yielding acrylate and the climate-active gas dimethylsulfide (DMS). SAR11 bacteria are the most abundant clade of heterotrophic bacteria in the oceans and they play a key role in DMSP catabolism. An important environmental factor affecting DMS generation via DMSP lyases is the availability of metal ions since they are essential cofactors for many of these enzymes. Here we examine the structure and activity of DddK in the presence of various metal ions. We have established that DddK containing a double stranded β-helical motif utilizes various divalent metal ions as cofactors for catalytic activity. However, nickel, an abundant metal ion in marine environments, adopts a distorted octahedral coordination environment and conferred the highest DMSP lyase activity. Crystal structures of cofactor bound DddK reveal key metal ion binding and catalytic residues and provide the first rationalization for varying activities with different metal ions. The structures of DddK along with site-directed mutagenesis and UV-visible studies are consistent with Tyr 64 acting as a base to initiate the β-elimination reaction of DMSP. Our biochemical and structural studies provide a detailed understanding of DMS generation by one of the ocean’s most prolific bacterium

Eicosanoid signalling blockade protects middle-aged mice from severe COVID-19

Coronavirus disease 2019 (COVID-19) is especially severe in aged populations1. Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective, but vaccine efficacy is partly compromised by the emergence of SARS-CoV-2 variants with enhanced transmissibility2. The emergence of these variants emphasizes the need for further development of anti-SARS-CoV-2 therapies, especially for aged populations. Here we describe the isolation of highly virulent mouse-adapted viruses and use them to test a new therapeutic drug in infected aged animals. Many of the alterations observed in SARS-CoV-2 during mouse adaptation (positions 417, 484, 493, 498 and 501 of the spike protein) also arise in humans in variants of concern2. Their appearance during mouse adaptation indicates that immune pressure is not required for selection. For murine SARS, for which severity is also age dependent, elevated levels of an eicosanoid (prostaglandin D2 (PGD2)) and a phospholipase (phospholipase A2 group 2D (PLA2G2D)) contributed to poor outcomes in aged mice3,4. mRNA expression of PLA2G2D and prostaglandin D2 receptor (PTGDR), and production of PGD2 also increase with ageing and after SARS-CoV-2 infection in dendritic cells derived from human peripheral blood mononuclear cells. Using our mouse-adapted SARS-CoV-2, we show that middle-aged mice lacking expression of PTGDR or PLA2G2D are protected from severe disease. Furthermore, treatment with a PTGDR antagonist, asapiprant, protected aged mice from lethal infection. PTGDR antagonism is one of the first interventions in SARS-CoV-2-infected animals that specifically protects aged animals, suggesting that the PLA2G2D–PGD2/PTGDR pathway is a useful target for therapeutic interventions.This work is supported in part by grants from the National Institutes of Health USA (NIH; P01 AI060699 (S.P. and P.B.M.) and R01 AI129269 (S.P.)) and BIOAGE Labs (S.P.). The Pathology Core is partially supported by the Center for Gene Therapy for Cystic Fibrosis (NIH grant P30 DK-54759) and the Cystic Fibrosis Foundation. P.B.M. is supported by the Roy J. Carver Charitable Trust. L.-Y.R.W. is supported by Mechanism of Parasitism Training Grant (T32 AI007511). We thank M. Gelb (University of Washington) for Pla2g2d−/− mice.Peer reviewe

<i>Bacillus anthracis</i> Prolyl 4‑Hydroxylase Interacts with and Modifies Elongation Factor Tu

Prolyl hydroxylation is a very common post-translational modification and plays many roles in eukaryotes such as collagen stabilization, hypoxia sensing, and controlling protein transcription and translation. There is a growing body of evidence that suggests that prokaryotes contain prolyl 4-hydroxylases (P4Hs) homologous to the hypoxia-inducible factor (HIF) prolyl hydroxylase domain (PHD) enzymes that act on elongation factor Tu (EFTu) and are likely involved in the regulation of bacterial translation. Recent biochemical and structural studies with a PHD from <i>Pseudomonas putida</i> (PPHD) determined that it forms a complex with EFTu and hydroxylates a prolyl residue of EFTu. Moreover, while animal, plant, and viral P4Hs act on peptidyl proline, most prokaryotic P4Hs have been known to target free l-proline; the exceptions include PPHD and a P4H from <i>Bacillus anthracis</i> (BaP4H) that modifies collagen-like proline-rich peptides. Here we use biophysical and mass spectrometric methods to demonstrate that BaP4H recognizes full-length BaEFTu and a BaEFTu 9-mer peptide for site-specific proline hydroxylation. Using size-exclusion chromatography coupled small-angle X-ray scattering (SEC–SAXS) and binding studies, we determined that BaP4H forms a 1:1 heterodimeric complex with BaEFTu. The SEC–SAXS studies reveal dissociation of BaP4H dimeric subunits upon interaction with BaEFTu. While BaP4H is unusual within bacteria in that it is structurally and functionally similar to the animal PHDs and collagen P4Hs, respectively, this work provides further evidence of its promiscuous substrate recognition. It is possible that the enzyme might have evolved to hydroxylate a universally conserved protein in prokaryotes, similar to the PHDs, and implies a functional role in <i>B. anthracis</i>

The pH dependence of DddW lyase activity.

<p>The optimal pH was determined by comparing the initial velocities (V_i) 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(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

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 (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

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

Metal binding affinities (K_d) of DddW.

<p>Titration of apo-DddW with increasing concentrations of the metal ions Fe(NH₄)₂(SO₄)₂ (added anaerobically), FeCl₃, CoCl₂, MnCl₂, CuCl₂ or NiCl₂ 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_d 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