Nitrite, previously considered physiologically irrelevant and a simple end product of endogenous nitric oxide (NO) metabolism, is now envisaged as a reservoir of NO to be activated in response to ...oxygen (O(2)) depletion. In the first part of this review, we summarize and compare the mechanisms of nitrite-dependent production of NO in selected bacteria and in eukaryotes. Bacterial nitrite reductases, which are copper or heme-containing enzymes, play an important role in the adaptation of pathogens to O(2) limitation and enable microrganisms to survive in the human body. In mammals, reduction of nitrite to NO under hypoxic conditions is carried out in tissues and blood by an array of metalloproteins, including heme-containing proteins and molybdenum enzymes. In humans, tissues play a more important role in nitrite reduction, not only because most tissues produce more NO than blood, but also because deoxyhemoglobin efficiently scavenges NO in blood. In the second part of the review, we outline the significance of nitrite in human health and disease and describe the recent advances and pitfalls of nitrite-based therapy, with special attention to its application in cardiovascular disorders, inflammation, and anti-bacterial defence. It can be concluded that nitrite (as well as nitrate-rich diet for long-term applications) may hold promise as therapeutic agent in vascular dysfunction and ischemic injury, as well as an effective compound able to promote angiogenesis.
Aspergillus fumigatus is a saprophytic ubiquitous fungus whose spores can trigger reactions such as allergic bronchopulmonary aspergillosis or the fatal invasive pulmonary aspergillosis. To survive ...in the lungs, the fungus must adapt to a hypoxic and nutritionally restrictive environment, exploiting the limited availability of aromatic amino acids (AAAs) in the best possible way, as mammals do not synthesize them. A key enzyme for AAAs catabolism in A. fumigatus is AroH, a pyridoxal 5′‐phosphate‐dependent aromatic aminotransferase. AroH was recently shown to display a broad substrate specificity, accepting L‐kynurenine and α‐aminoadipate as amino donors besides AAAs. Given its pivotal role in the adaptability of the fungus to nutrient conditions, AroH represents a potential target for the development of innovative therapies against A. fumigatus‐related diseases. We have solved the crystal structure of Af‐AroH at 2.4 Å resolution and gained new insight into the dynamics of the enzyme's active site, which appears to be crucial for the design of inhibitors. The conformational plasticity of the active site pocket is probably linked to the wide substrate specificity of AroH.
The cytosolic and mitochondrial isoforms of serine hydroxymethyltransferase (SHMT1 and SHMT2, respectively) are well-recognized targets of cancer research, since their activity is critical for purine ...and pyrimidine biosynthesis and because of their prominent role in the metabolic reprogramming of cancer cells. Here we show that 3-bromopyruvate (3BP), a potent novel anti-tumour agent believed to function primarily by blocking energy metabolism, differentially inactivates human SHMT1 and SHMT2. SHMT1 is completely inhibited by 3BP, whereas SHMT2 retains a significant fraction of activity. Site directed mutagenesis experiments on SHMT1 demonstrate that selective inhibition relies on the presence of a cysteine residue at the active site of SHMT1 (Cys204) that is absent in SHMT2. Our results show that 3BP binds to SHMT1 active site, forming an enzyme-3BP complex, before reacting with Cys204. The physiological substrate l-serine is still able to bind at the active site of the inhibited enzyme, although catalysis does not occur. Modelling studies suggest that alkylation of Cys204 prevents a productive binding of l-serine, hampering interaction between substrate and Arg402. Conversely, the partial inactivation of SHMT2 takes place without the formation of a 3BP-enzyme complex. The introduction of a cysteine residue in the active site of SHMT2 by site directed mutagenesis (A206C mutation), at a location corresponding to that of Cys204 in SHMT1, yields an enzyme that forms a 3BP-enzyme complex and is completely inactivated. This work sets the basis for the development of selective SHMT1 inhibitors that target Cys204, starting from the structure and reactivity of 3BP.
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•3-Bromopyruvate completely inhibits SHMT1, alkylating the active site Cys204.•SHMT2, which lacks Cys 204, is only partially inhibited by 3-bromopyruvate.•3-Bromopyruvate binds to SHMT1 active site forming a non-covalent complex.•Introducing a Cys residue in SHMT2 active site yields a complete inhibition.•3-Bromopyruvate is a lead compound for the design of selective SHMT1 inhibitors.
Background/Aim: The connection between prostate cancer and inflammation has been proposed many years ago, but very little is known about the metabolic adaptations of prostate cells in case of ...infection or inflammation. The aim of this study was to examine the effect of the stimulation of Toll-like receptor 3 (TLR3) on the metabolism of prostate cancer (PCa) cell lines and benign prostate cells. Materials and Methods: Cytofluorimetry, qRT-PCR, western blot and Gas-chromatography/Mass-spectrometry were used. Results: Reprogramming of glucose utilization involving hypoxia-inducible factor 1-alpha (HIF-1α) and the extracellular adenosine axis was observed. TLR3 stimulation synergized with adenosine receptor A2b on PCa cells, and induced a strong production of lactate, exacerbating the Warburg effect. Moreover, stimulation of benign prostate cells with poly I:C reduced lactate secretion, a characteristic typical of the neoplastic transformation. Conclusion: TLR3 stimulation promotes metabolic adaptations likely involved in the mechanisms of disease onset and progression.
Cancer cells reprogramme one‐carbon metabolism (OCM) to sustain growth and proliferation. Depending on cell demands, serine hydroxymethyltransferase (SHMT) dynamically changes the fluxes of OCM by ...reversibly converting serine and tetrahydrofolate (THF) into 5,10‐methylene‐THF and glycine. SHMT is a tetrameric enzyme that mainly exists in three isoforms; two localize in the cytosol (SHMT1/SHMT2α) and one (SHMT2) in the mitochondria. Both the cytosolic isoforms can also translocate to the nucleus to sustain de novo thymidylate synthesis and support cell proliferation. Finally, the expression levels of the different isoforms are regulated to a certain extent by a yet unknown crosstalk mechanism. We have designed and fully characterized a set of three SHMT1 mutants, which uncouple the oligomeric state of the enzyme from its catalytic activity. We have then investigated the effects of the mutations on SHMT1 nuclear localization, cell viability and crosstalk in lung cancer cells (A549; H1299). Our data reveal that in these cell lines de novo thymidylate synthesis requires SHMT1 to be active, regardless of its oligomeric state. We have also confirmed that the crosstalk between the cytosolic and mitochondrial SHMT actually takes place and regulates the expression of the two isoforms. Apparently, the crosstalk mechanism is independent from the oligomeric state and the catalytic activity of SHMT1.
Database
Structural data are available in the PDB under the accession number 6FL5
We designed a set of three serine hydroxymethyltransferase 1 (SHMT1) mutants to uncouple the oligomeric state of the enzyme from its catalytic activity. We investigated the effects of these mutations on nuclear localization, cell viability, and crosstalk between SHMT1 and SHMT2 in lung cancer cells. In these cell lines, de novo thymidylate synthesis requires SHMT1 to be active regardless of its oligomeric state.
1 Department of Biochemical Sciences A. Rossi Fanelli, Sapienza University of Rome, Rome, Italy
2 Consorzio INBB, 00136 Rome, Italy
Pseudomonas aeruginosa is a well-known pathogen in chronic ...respiratory diseases such as cystic fibrosis. Infectivity of P. aeruginosa is related to the ability to grow under oxygen-limited conditions using the anaerobic metabolism of denitrification, in which nitrate is reduced to dinitrogen via nitric oxide (NO). Denitrification is activated by a cascade of redox-sensitive transcription factors, among which is the DNR regulator, sensitive to nitrogen oxides. To gain further insight into the mechanism of NO-sensing by DNR, we have developed an Escherichia coli -based reporter system to investigate different aspects of DNR activity. In E. coli DNR responds to NO, as shown by its ability to transactivate the P. aeruginosa norCB promoter. The direct binding of DNR to the target DNA is required, since mutations in the helix–turn–helix domain of DNR and specific nucleotide substitutions in the consensus sequence of the norCB promoter abolish the transcriptional activity. Using an E. coli strain deficient in haem biosynthesis, we have also confirmed that haem is required in vivo for the NO-dependent DNR activity, in agreement with the property of DNR to bind haem in vitro . Finally, we have shown, we believe for the first time, that DNR is able to discriminate in vivo between different diatomic signal molecules, NO and CO, both ligands of the reduced haem iron in vitro , suggesting that DNR responds specifically to NO.
Correspondence Francesca Cutruzzolà francesca.cutruzzola{at}uniroma1.it
Abbreviations: ALA, 5-aminolaevulinic acid; CRP, cAMP receptor protein; DNR, dissimilatory nitrate respiration regulator; FNR, fumarate and nitrate reductase regulator; HTH, helix–turn–helix; SNP, sodium nitroprusside; β -gal, β -galactosidase
The alanine:glyoxylate aminotransferase (AGT), a hepatocyte-specific pyridoxal-5'-phosphate (PLP) dependent enzyme, transaminates L-alanine and glyoxylate to glycine and pyruvate, thus detoxifying ...glyoxylate and preventing pathological oxalate precipitation in tissues. In the widely accepted catalytic mechanism of the aminotransferase family, the lysine binding to PLP acts as a catalyst in the stepwise 1,3-proton transfer, interconverting the external aldimine to ketimine. This step requires protonation by a conserved aspartate of the pyridine nitrogen of PLP to enhance its ability to stabilize the carbanionic intermediate. The aspartate residue is also responsible for a significant geometrical distortion of the internal aldimine, crucial for catalysis. We present the structure of human AGT in which complete X-ray photoreduction of the Schiff base has occurred. This result, together with two crystal structures of the conserved aspartate pathogenic variant (D183N) and the molecular modeling of the transaldimination step, led us to propose that an interplay of opposite forces, which we named spring mechanism, finely tunes PLP geometry during catalysis and is essential to move the external aldimine in the correct position in order for the 1,3-proton transfer to occur.
Bacteria react to adverse environmental stimuli by clustering into organized communities called biofilms. A remarkably sophisticated control system based on the dinucleotide 3'-5' cyclic diguanylic ...acid (c-di-GMP) is involved in deciding whether to form or abandon biofilms. The ability of c-di-GMP to form self-intercalated dimers is also thought to play a role in this complex regulation. A great advantage in the quest of elucidating the catalytic properties of the enzymes involved in c-di-GMP turnover (diguanylate cyclases and phosphodiesterases) would come from the availability of an experimental approach for in vitro quantification of c-di-GMP in real-time. Here, we show that c-di-GMP can be detected and quantified by circular dichroism (CD) spectroscopy in the low micromolar range. The method is based on the selective ability of manganese ions to induce formation of the intercalated dimer of the c-di-GMP dinucleotide in solution, which displays an intense sigmoidal CD spectrum in the near-ultraviolet region. This characteristic spectrum originates from the stacking interaction of the four mutually intercalated guanines, as it is absent in the other cyclic dinucleotide 3'-5' cyclic adenilic acid (c-di-AMP). Thus, near-ultraviolet CD can be used to effectively quantify in real-time the activity of diguanylate cyclases and phosphodiesterases in solution.
cd1 nitrite reductase (NIR) is a key enzyme in the denitrification process that reduces nitrite to nitric oxide (NO). It contains a specialized d1-heme cofactor, found only in this class of enzymes, ...where the substrate, nitrite, binds and is converted to NO. For a long time, it was believed that NO must be released from the ferric d1-heme to avoid enzyme inhibition by the formation of ferrous−nitroso complex, which was considered as a dead-end product. However, recently an enhanced rate of NO dissociation from the ferrous form, not observed in standard b-type hemes, has been reported and attributed to the unique d1-heme structure (Rinaldo, S.; Arcovito, A.; Brunori, M.; Cutruzzolà, F. J. Biol. Chem. 2007, 282, 14761−14767). Here, we report on a detailed study of the spatial and electronic structure of the ferrous d1-heme NO complex from Pseudomonas aeruginosa cd1 NIR and two mutants Y10F and H369A/H327A in solution, searching for the unique properties that are responsible for the relatively fast release. There are three residues at the “distal” side of the heme (Tyr10, His327, and His369), and in this work we focus on the identification and characterization of possible H-bonds they can form with the NO, thereby affecting the stability of the complex. For this purpose, we have used high field pulse electron−nuclear double resonance (ENDOR) combined with density functional theory (DFT) calculations. The DFT calculations were essential for assigning and interpreting the ENDOR spectra in terms of geometric structure. We have shown that the NO in the nitrosyl d1-heme complex of cd1 NIR forms H-bonds with Tyr10 and His369, whereas the second conserved histidine, His327, appears to be less involved in NO H-bonding. This is in contrast to the crystal structure that shows that Tyr10 is removed from the NO. We have also observed a larger solvent accessibility to the distal pocket in the mutants as compared to the wild-type. Moreover, it was shown that the H-bonding network within the active site is dynamic and that a change in the protonation state of one of the residues does affect the strength and position of the H-bonds formed by the others. In the Y10F mutant, His369 is closer to the NO, whereas mutation of both distal histidines displaces Tyr10, removing its H-bond. The implications of the H-bonding network found in terms of the complex stability and catalysis are discussed.
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