This is a collection of invited talks and oral contributions presented by the leading scientists in their fields, summarizing the most recent progress of, and new prospects for, nuclear physics ...research.It covers a broad range of the recent developments in nuclear physics: reactions between massive nuclei leading to superheavy element formation; radioactive beams and neutron-rich systems; exotic nuclei and nuclear astrophysics; new states of nuclear matter.
Among the over 50 gyrate atrophy‐causing mutations of ornithine δ‐aminotransferase (OAT), the R180T involves an active site residue located at the dimer interface, which in the crystal structure of ...OAT complexed with 5‐fluoromethylornithine engages a salt bridge with the α‐carboxylate of the substrate analogue. Starting from the previous finding that no transaminase activity was detected in CHO‐K1 cells expressing the R180T variant, here we try to shed light at the protein level on the structural and/or functional defects of the R180T variant. To this aim, the variant has been cloned, expressed, purified and characterized by a combination of biochemical and structural studies. Although the R180T variant shares a similar overall conformation with the wild‐type, its crystal structure solved at 1.8 Ǻ reveals slight structural alterations at the active site and at the dimeric interface. These changes are consistent with the spectroscopic and kinetic results, indicating that the variant, as compared with the wild‐type OAT, shows (a) an increased Km value for l‐ornithine (l‐Orn), (b) an altered pyridoxal 5′‐phosphate binding mode and affinity and (c) an increased thermostability. In addition, the R180T mutant exhibits a remarkable loss of catalytic activity and is endowed with the ability to catalyse not only the δ‐transamination but also, albeit to a lesser extent, the α‐transamination of l‐Orn. Overall, these data indicate that the slight structural changes caused by the R180T mutation, preventing a proper collocation of l‐Orn at the active site of OAT, are responsible for the notable reduction of the catalytic efficiency.
Enzymes
Ornithine aminotransferase EC 2.6.1.13.
Databases
6HX7.pdb.
The R180T variant of human ornithine δ‐aminotransferase (OAT) associated with gyrate atrophy. A multidisciplinary approach including biochemical, computational, X‐ray and NMR analyses revealed that R180T mutation causes a remarkable reduction of the catalytic efficiency of the OAT. Moreover, R180T variant resulted able to catalyse not only the δ‐transamination but also the α‐transamination of l‐ornithine indicating that Arg180 contributes to the reaction specificity of the human OAT.
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.
Serine hydroxymethyltransferase (SHMT) is a pivotal enzyme in one-carbon metabolism that catalyses the reversible conversion of serine and tetrahydrofolate into glycine and methylenetetrahydrofolate. ...It exists in cytosolic (SHMT1) and mitochondrial (SHMT2) isoforms. Research on one-carbon metabolism in cancer cell lines has shown that SHMT1 preferentially catalyses serine synthesis, whereas in mitochondria SHMT2 is involved in serine breakdown. Recent research has focused on the identification of inhibitors that bind at the folate pocket. We have previously found that a representative derivative of the pyrazolopyran scaffold, namely 2.12, inhibits both SHMT isoforms, with a preference for SHMT1, causing apoptosis in lung cancer cell lines. Here we show that the affinity of 2.12 for SHMT depends on the identity of the amino acid substrate bound to the enzyme. The dissociation constant of 2.12 is 50-fold lower when it binds to SHMT1 enzyme-serine complex, as compared to the enzyme-glycine complex. Evidence is presented for a similar behaviour of compound 2.12 in the cellular environment. These findings suggest that the presence and identity of the amino acid substrate should be considered when designing SHMT inhibitors. Moreover, our data provide the proof-of-concept that SHMT inhibitors selectively targeting the directionality of one-carbon metabolism flux could be designed.
•The 2.12 inhibitor binds with higher affinity to SHMT-Ser than to SHMT-Gly complex.•Differential affinity probably depends on a hydrogen bond between Ser and 2.12.•Preference of 2.12 for SHMT-Ser is also evident in live cells.
The reduction of nitrite into nitric oxide (NO) in denitrifying bacteria is catalyzed by nitrite reductase. In several species, this enzyme is a heme-containing protein with one c heme and one d1 ...heme per monomer (cd1NiR), encoded by the nirS gene. For many years, the evidence of a link between NO and this hemeprotein represented a paradox, given that NO was known to tightly bind and, possibly, inhibit hemeproteins, including cd1NiRs. It is now established that, during catalysis, cd1NiRs diverge from "canonical" hemeproteins, since the product NO rapidly dissociates from the ferrous d1 heme, which, in turn, displays a peculiar "low" affinity for NO (KD=0.11 μM at pH 7.0). It has been also previously shown that the c heme reacts with NO at acidic pH but c heme nitrosylation was not extensively investigated, given that in cd1NiR it was considered a side reaction, rather than a genuine process controlling catalysis. The spectroscopic study of the reaction of cd1NiR and its semi-apo derivative (containing the sole c heme) with NO reported here shows that c heme nitrosylation is enhanced during catalysis; this evidence has been discussed in order to assess the potential of c heme nitrosylation as a regulatory process, as observed for cytochrome c nitrosylation in mammalian mitochondria.
Cyclic di-GMP (c-di-GMP) is a widespread second messenger that plays a key role in bacterial biofilm formation. The compound’s ability to assume multiple conformations allows it to interact with a ...diverse set of target macromolecules. Here, we analyzed the binding mode of c-di-GMP to the allosteric inhibitory site (I-site) of diguanylate cyclases (DGCs) and compared it to the conformation adopted in the catalytic site of the EAL phosphodiesterases (PDEs). An array of novel molecules has been designed and synthesized by simplifying the native c-di-GMP structure and replacing the charged phosphodiester backbone with an isosteric nonhydrolyzable 1,2,3-triazole moiety. We developed the first neutral small molecule able to selectively target DGCs discriminating between the I-site of DGCs and the active site of PDEs; this molecule represents a novel tool for mechanistic studies, particularly on those proteins bearing both DGC and PDE modules, and for future optimization studies to target DGCs in vivo.
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► The thioredoxin system is crucial to the viability of Plasmodium falciparum. ► The system is headed by NADPH-dependent thioredoxin reductase (PfTrxR). ► We present the first crystal ...structure of PfTrxR, a validated drug target. ► The interface and the central cavity are different from the human counterpart. ► These differences could be exploited for future rational inhibitors design.
Plasmodium falciparum is the vector of the most prevalent and deadly form of malaria, and, among the Plasmodium species, it is the one with the highest rate of drug resistance. At the basis of a rational drug design project there is the selection and characterization of suitable target(s). Thioredoxin reductase, the first protection against reactive oxygen species in the erythrocytic phase of the parasite, is essential for its survival. Hence it represents a good target for the design of new anti-malarial active compounds. In this paper we present the first crystal structure of recombinant P. falciparum thioredoxin reductase (PfTrxR) at 2.9Å and discuss its differences with respect to the human orthologue. The most important one resides in the dimer interface, which offers a good binding site for selective non competitive inhibitors. The striking conservation of this feature among the Plasmodium parasites, but not among other Apicomplexa parasites neither in mammals, boosts its exploitability.
The aim of the Symposium has been to widen and detail the discussion of problems arising in front of experimental and theoretical groups, and to find overlap between different approaches and methods ...which are devoted to the studying dynamics of nuclear reactions. Therefore, the reaction product yields are determined by various processes in competition. The main topics of the Symposium have been devoted to the following well sounded problems of nuclear reactions: The synthesis of superheavy elements and the study of exotic nuclei far from the valley of the beta stability. The production mechanism of the observed new elements and isotopes. The study of transfer reactions as a way to understand mechanism of evolution of from the deep-inelastic collisions to fusion regime. The study of non-equilibrium stage of the reaction mechanism and distribution of the excitation energy between binary reaction products including spontaneous fission products are still important to have a correct presentation about the whole reaction mechanism. The similarities and difference between fusion–fission and quasifission products. Unambiguity in estimation of the realistic fusion cross sections by the experimental and theoretical methods. Angular anisotropy of the complete and incomplete fusion reaction products. The effect of the nuclear shell structure in formation of the mass symmetric and asymmetric fission products. The investigation of the role of angular momentum, mass asymmetry and orientation angles of the symmetry axes of colliding nuclei in the entrance channel in formation of the evaporation residues, mass and angular distribution of the fusion–fission and quasifission products. Multi-fragmentation and symmetry energy. The new experimental and theoretical investigations on these and related topics allow researchers to improve knowledge about nucleus–nucleus interaction dynamics and to make conclusions about perspectives in the study of the landscape of islands superheavy elements and exotic nuclei far from the valley of the beta stability. Giorgio Giardina Avazbek K Nasirov Giuseppe Mandaglio Editors of the Proceedings of ECHIC2013
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