Genes for asparagine metabolism in Lotus japonicus : differential expression and interconnection with photorespiration

Background: Asparagine is a very important nitrogen transport and storage compound in plants due to its high nitrogen/carbon ratio and stability. Asparagine intracellu lar concentration depends on a balance between asparagine biosynthesis and degradation. The main enzymes involved in asparagine metabolism are as paragine synthetase (ASN), asparaginase (NSE) and serine-glyoxylate aminotransfera se (SGAT). The study of the genes encoding for these enzymes in the model legume Lotus japonicus is of particular interest since it has been proposed that asparagine is the principal molecule used to transport reduced nitrogen within the plant in most temperate legumes. Results: A differential expression of genes encoding for seve ral enzymes involved in asparagine metabolism was detected in L. japonicus . ASN is encoded by three genes, LjASN1 was the most highly expressed in mature leaves while LjASN2 expression was negligible and LjASN3 showed a low expression in this organ, suggesting that LjASN1 is the main gene responsible for asparagine synthesis in mature leaves. In young leaves, LjASN3 was the only ASN gene expressed although at low levels, while all the three genes encoding for NSE were highly expressed, especially LjNSE1 .Innodules, LjASN2 and LjNSE2 were the most highly expressed genes, suggesting an important role for these genes in this organ. Several lines of evidence support the connection between asparagine metabolic genes and photorespiration in L. japonicus : a) a mutant plant deficient in LjNSE1 showed a dramatic decrease in the expression of the two genes encoding for SGAT; b) expression of the genes involved in asparagine metabolism is altered in a photorespiratory mutant lacking plastidic glutamine synthetase; c) a clustering analysis indicated a similar pattern of expression among several genes involved in photorespiratory and asparagine metabolism, indicating a clear link between LjASN1 and LjSGAT genes and photorespiration. Conclusions: The results obtained in this paper indicate the exis tence of a differential expression of asparagine metabolic genes in L. japonicus and point out the crucial relevance of particular genes in different organs. Moreover, the data presented establish clear links betw een asparagine and photorespiratory metabolic genes in this plant.Junta de Andalucía (P10-CVI- 6368)FEDER-Ministerio de Economía y Competitividad (AGL 2014 – 54413-R

Local conformational changes in the 8–17 deoxyribozyme core induced by activating and inactivating divalent metal ions

The 8-17 deoxyribozyme (DNAzyme) is a catalytic DNA molecule capable of cleaving specific RNA substrates. The deoxyribozyme is activated by a wide variety of divalent metal ions, from Mg2+ to Pb2+, with just a few exceptions. It is not clear if metal ions are directly involved in catalysis, or are required to attain an active conformation, or both. In particular, the connection between metal-induced global structural rearrangements and catalysis is not straightforward. To gain more information on the local structural changes induced by metal ions, we introduced fluorescent 2-aminopurine (2-Ap) residues at different positions of the 8-17 'core'. We found that a construct containing 2-Ap at position 15 was best suited to monitor conformational changes in the presence of Mg2+, Ca2+ or Mn2+. Binding of these activating metal ions caused a local rearrangement at position 15, apparently entailing decreased stacking of the 2-Ap base. The metal dependence for such conformational change was generally hyperbolic (suggesting it mirrored the binding by a single metal ion) and yielded apparent dissociation constants close to those required for activation. In contrast, Cu2+, a divalent metal ion which does not support catalysis, caused in the deoxyribozyme a slow, reversible inactivation, which correlated with a very distinct conformational change at position 15

Kinetic and thermodynamic characterization of the RNA-cleaving 8-17 deoxyribozyme

The 8-17 deoxyribozyme is a small DNA catalyst of significant applicative interest. We have analyzed the kinetic features of a well behaved 8-17 construct and determined the influence of several reaction conditions on such features, providing a basis for further exploration of the deoxyribozyme mechanism. The 8-17 bound its substrate with a rate constant ∼10-fold lower than those typical for the annealing of short complementary oligonucleotides. The observed free energy of substrate binding indicates that an energetic penalty near to +7 kcal/mol is attributable to the deoxyribozyme core. Substrate cleavage required divalent metal ion cofactors, and the dependence of activity on the concentration of Mg(2+), Ca(2+) or Mn(2+) suggests the occurrence of a single, low-specificity binding site for activating ions. The efficiency of activation correlated with the Lewis acidity of the ion cofactor, compatible with a metal-assisted deprotonation of the reactive 2′-hydroxyl group. However, alternative roles of the metal ions cannot be excluded, because those ions that are stronger Lewis acids are also capable of forming stronger interactions with ligands such as the phosphate oxygens. The apparent enthalpy of activation for the 8-17 reaction was close to the values observed for hydroxide-catalyzed and hammerhead ribozyme-catalyzed RNA cleavage

Heme uptake in bacterial pathogens

Iron is an essential nutrient for the survival of organisms. Bacterial pathogens possess specialized pathways to acquire heme from their human hosts. In this review, we present recent structural and biochemical data that provide mechanistic insights into several bacterial heme uptake pathways, encompassing the sequestration of heme from human hemoproteins to secreted or membrane-associated bacterial proteins, the transport of heme across bacterial membranes, and the degradation of heme within the bacterial cytosol to liberate iron. The pathways for heme transport into the bacterial cytosol are divergent, harboring non-homologous protein sequences, novel structures, varying numbers of proteins, and different mechanisms. Congruously, the breakdown of heme within the bacterial cytosol by sequence-divergent proteins releases iron and distinct degradation products

Glutamine Synthetase in Legumes: Recent Advances in Enzyme Structure and Functional Genomics

Abstract: Glutamine synthetase (GS) is the key enzyme involved in the assimilation of ammonia derived either from nitrate reduction, N2 fixation, photorespiration or asparagine breakdown. A small gene family is encoding for different cytosolic (GS1) or plastidic (GS2) isoforms in legumes. We summarize here the recent advances carried out concerning the quaternary structure of GS, as well as the functional relationship existing between GS2 and processes such as nodulation, photorespiration and water stress, in this latter case by means of proline production. Functional genomic analysis using GS2-minus mutant reveals the key role of GS2 in the metabolic control of the plants and, more particularly, in carbon metabolism

Additional file 2: Figure. S1. of Genes for asparagine metabolism in Lotus japonicus: differential expression and interconnection with photorespiration

Phylogenetic tree of asparagine synthetases. A dendrogram of asparagine synthetase sequences was generated by PILEUP as previously described [5] including LjASN1, LjASN2 and LjASN3 from L. japonicus. Class-I and class-II phylogenetic clades are shown. (TIFF 626 kb

Additional file 1: Table S1. of Genes for asparagine metabolism in Lotus japonicus: differential expression and interconnection with photorespiration

Sequences of primers used in qRT-PCR experiments. (DOC 35 kb

Additional file 3: Figure. S2. of Genes for asparagine metabolism in Lotus japonicus: differential expression and interconnection with photorespiration

Structures of the LjASN, LjNSE and LjSGAT genes from L. japonicus. Exons are represented as boxes. (TIFF 134 kb

Diversification of β-Augmentation Interactions between CDI Toxin/Immunity Proteins.

Contact-dependent growth inhibition (CDI) is a widespread mechanism of inter-bacterial competition mediated by the CdiB/CdiA family of two-partner secretion proteins. CdiA effectors carry diverse C-terminal toxin domains (CdiA-CT), which are delivered into neighboring target cells to inhibit growth. CDI(+) bacteria also produce CdiI immunity proteins that bind specifically to cognate CdiA-CT toxins and protect the cell from auto-inhibition. Here, we compare the structures of homologous CdiA-CT/CdiI complexes from Escherichia coli EC869 and Yersinia pseudotuberculosis YPIII to explore the evolution of CDI toxin/immunity protein interactions. Both complexes share an unusual β-augmentation interaction, in which the toxin domain extends a β-hairpin into the immunity protein to complete a six-stranded anti-parallel sheet. However, the specific contacts differ substantially between the two complexes. The EC869 β-hairpin interacts mainly through direct H-bond and ion-pair interactions, whereas the YPIII β-hairpin pocket contains more hydrophobic contacts and a network of bridging water molecules. In accord with these differences, we find that each CdiI protein only protects target bacteria from its cognate CdiA-CT toxin. The compact β-hairpin binding pocket within the immunity protein represents a tractable system for the rationale design of small molecules to block CdiA-CT/CdiI complex formation. We synthesized a macrocyclic peptide mimic of the β-hairpin from EC869 toxin and solved its structure in complex with cognate immunity protein. These latter studies suggest that small molecules could potentially be used to disrupt CDI toxin/immunity complexes

Additional file 4: Table S2. of Genes for asparagine metabolism in Lotus japonicus: differential expression and interconnection with photorespiration

List and description of the L. japonicus transcriptomic data analyzed in the present work. All transcriptomic data are available online in the Arrayexpress database. (XLSX 14 kb