Putrescine Detected in Strains of \u3ci\u3eStaphylococcus aureus\u3c/i\u3e

Most forms of life, including the archaea, bacteria, and eukaryotes synthesize the polyamine putrescine. Although putrescine is widely distributed, several Gram-positive bacteria, including Staphylococcus aureus (S. aureus), appear to be the exceptions. We report here that strains of S. aureus can produce the polyamine putrescine, as well as the derivative N-acetyl-putrescine. Three strains of S. aureus from the American Type Culture Collection (ATCC), one strain listed in the National Center for Biotechnology Information (NCBI) database, whose genomic sequence is well defined, and well as eight strains from S. aureus-induced brain abscesses of individual patients from multiple geographic locations were evaluated. Each strain was grown in complete chemically defined medium (CDM) under stringent conditions, after which the partially purified conditioned medium (CM) was analyzed by mass spectroscopy (MS), and the data were reported as the ratio of experimental results to controls. We confirmed the synthesis of putrescine by S. aureus by using 13C/15N-labeled arginine as a tracer. We found that agmatine, N-acetyl-putrescine, ornithine, citrulline, proline, and NH3 were all labeled with heavy isotope derived from 13C/15N-labeled arginine. None of the strains examined produced spermine or spermidine, but strains from either ATCC or human brain abscesses produced putrescine and/or its derivative N-acetyl-putrescine

13C NMR Characterization of an Exchange Reaction between CO and CO2 Catalyzed by Carbon Monoxide Dehydrogenase†

ABSTRACT: Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel-iron-sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s-1 at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the 13CO2 line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between 12CO and 13CO2 in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of 13CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN-) was also probed by 13C NMR. A functional C-cluster is not required fo

Coordination of metal center biogenesis in human cytochrome c oxidase

Mitochondrial cytochrome c oxidase (CcO) or respiratory chain complex IV is a heme aa3- copper oxygen reductase containing metal centers essential for holo-complex biogenesis and enzymatic function that are assembled by subunit-specific metallochaperones. The enzyme has two copper sites located in the catalytic core subunits. The COX1 subunit harbors the CuB site that tightly associates with heme a3 while the COX2 subunit contains the binuclear CuA site. Here, we report that in human cells the CcO copper chaperones form macromolecular assemblies and cooperate with several twin CX9C proteins to control heme a biosynthesis and coordinate copper transfer sequentially to the CuA and CuB sites. These data on CcO illustrate a mechanism that regulates the biogenesis of macromolecular enzymatic assemblies with several catalytic metal redox centers and prevents the accumulation of cytotoxic reactive assembly intermediates

Cadmium and Secondary Structure-dependent Function of a Degron in the Pca1p Cadmium Exporter

Protein turnover is a critical cellular process regulating biochemical pathways and destroying terminally misfolded or damaged proteins. Pca1p, a cadmium exporter in the yeast Saccharomyces cerevisiae, is rapidly degraded by the endoplasmic reticulum-associated degradation (ERAD) system via a cis-acting degron that exists at the 250–350 amino acid region of Pca1p and is transferable to other proteins to serve as a degradation signal. Cadmium stabilizes Pca1p in a manner dependent on the degron. This suggested that cadmium-mediated masking of the degron impedes its interaction with the molecular factors involved in the ERAD. The characteristics and mechanisms of action of the degron in Pca1p and most of those in other proteins however remain to be determined. The results presented here indicate that specific cysteine residues in a degron of Pca1p sense cadmium.Anunbiased approach selecting non-functional degrons indicated a critical role of hydrophobic amino acids in the degron for its function.Asecondary structure modeling predicted the formation of an amphipathic helix. Site-directed mutagenesis confirmed the functional significance of the hydrophobic patch. Last, hydrophobic amino acids in the degron- and cadmium-binding region affected the interaction of Pca1p with the Ssa1p molecular chaperone, which is involved in ERAD. These results reveal the mechanism of action of the degron, which might be useful for the identification and characterization of other degrons

Cobalt-sulfur coordination chemistry drives B\u3csub\u3e12\u3c/sub\u3e loading onto methionine synthase

Cobalt-sulfur (Co-S) coordination is labile to both oxidation and reduction chemistry and is rarely seen in Nature. Cobalamin (or vitamin B12) is an essential cobalt-containing organometallic cofactor in mammals, and is escorted via an intricate network of chaperones to a single cytoplasmic target, methionine synthase. In this study, we report that the human cobalamin trafficking protein, MMADHC, exploits the chemical lability of Co-S coordination, for cofactor off-loading onto methionine synthase. Cys-261 on MMADHC serves as the -axial ligand to cobalamin. Complex formation between MMADHC and methionine synthase is signaled by loss of the lower axial nitrogen ligand, leading to five-coordinate thiolato-cobalamin. Nucleophilic displacement by the vicinal thiolate, Cys-262, completes cofactor transfer to methionine synthase and release of a cysteine disulfide-containing MMADHC. The physiological relevance of this mechanism is supported by clinical variants of MMADHC, which impair cofactor binding and off-loading, explaining the molecular basis of the associated homocystinuria

Functional Impact of a Cancer-Related Variant in Human Δ\u3csup\u3e1\u3c/sup\u3e‑Pyrroline-5-Carboxylate Reductase 1

Pyrroline-5-carboxylate reductase (PYCR) is a proline biosynthetic enzyme that catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline. Humans have three PYCR isoforms, with PYCR1 often upregulated in different types of cancers. Here, we studied the biochemical and structural properties of the Thr171Met variant of PYCR1, which is found in patients with malignant melanoma and lung adenocarcinoma. Although PYCR1 is strongly associated with cancer progression, characterization of a PYCR1 variant in cancer patients has not yet been reported. Thr171 is conserved in all three PYCR isozymes and is located near the P5C substrate binding site. We found that the amino acid replacement does not affect thermostability but has a profound effect on PYCR1 catalytic activity. The kcat of the PYCR1 variant T171M is 100- to 200-fold lower than wild-type PYCR1 when P5C is the variable substrate, and 10- to 25-fold lower when NAD(P)H is varied. A 1.84 Å resolution X-ray crystal structure of T171M reveals that the Met side chain invades the P5C substrate binding site, suggesting that the catalytic defect is due to steric clash preventing P5C from achieving the optimal pose for hydride transfer from NAD(P)H. These results suggest that any impact on PYCR1 function associated with T171M in cancer does not derive from increased catalytic activity

Structural and Mechanistic Insights into Hemoglobincatalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal

Structural and Mechanistic Insights into Hemoglobincatalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal

Structural and Mechanistic Insights into Hemoglobincatalyzed Hydrogen Sulfide Oxidation and the Fate of Polysulfide Products

Hydrogen sulfide is a cardioprotective signaling molecule but is toxic at elevated concentrations. Red blood cells can synthesize H2S but, lacking organelles, cannot dispose of H2S via the mitochondrial sulfide oxidation pathway. We have recently shown that at high sulfide concentrations, ferric hemoglobin oxidizes H2S to a mixture of thiosulfate and iron-bound polysulfides in which the latter species predominates. Here, we report the crystal structure of human hemoglobin containing low spin ferric sulfide, the first intermediate in heme-catalyzed sulfide oxidation. The structure provides molecular insights into why sulfide is susceptible to oxidation in human hemoglobin but is stabilized against it in HbI, a specialized sulfide-carrying hemoglobin from a mollusk adapted to life in a sulfide-rich environment. We have also captured a second sulfide bound at a postulated ligand entry/exit site in the α-subunit of hemoglobin, which, to the best of our knowledge, represents the first direct evidence for this site being used to access the heme iron. Hydrodisulfide, a postulated intermediate at the junction between thiosulfate and polysulfide formation, coordinates ferric hemoglobin and, in the presence of air, generated thiosulfate. At low sulfide/heme iron ratios, the product distribution between thiosulfate and iron-bound polysulfides was approximately equal. The iron-bound polysulfides were unstable at physiological glutathione concentrations and were reduced with concomitant formation of glutathione persulfide, glutathione disulfide, and H2S. Hence, although polysulfides are unlikely to be stable in the reducing intracellular milieu, glutathione persulfide could serve as a persulfide donor for protein persulfidation, a posttranslational modification by which H2S is postulated to signal

A 4-Selenocysteine, 2-Selenocysteine Insertion Sequence (SECIS) Element Methionine Sulfoxide Reductase from \u3ci\u3eMetridium senile\u3c/i\u3e Reveals a Non-catalytic Function of Selenocysteines

Selenocysteine (Sec) residues occur in thiol oxidoreductase families, and functionally characterized selenoenzymes typically have a single Sec residue used directly for redox catalysis. However, how new Sec residues evolve and whether non-catalytic Sec residues exist in proteins is not known. Here, we computationally identified several genes with multiple Sec insertion sequence (SECIS) elements, one of which was a methionine-Rsulfoxide reductase (MsrB) homolog from Metridium senile that has four in-frame UGA codons and two nearly identical SECIS elements. One of the UGA codons corresponded to the conserved catalytic Sec or Cys in MsrBs, whereas the three other UGA codons evolved recently and had no homologs with Sec or Cys in these positions. Metabolic 75Se labeling showed that all four in-frame UGA codons supported Sec insertion and that both SECIS elements were functional and collaborated in Sec insertion at each UGA codon. Interestingly, recombinant M. senile MsrB bound iron, and further analyses suggested the possibility of binding an iron-sulfur cluster by the protein. These data show that Sec residues may appear transiently in genes containing SECIS elements and be adapted for non-catalytic functions