Surprising arginine biosynthesis: a reappraisal of the enzymology and evolution of the pathway in microorganisms.

Major aspects of the pathway of de novo arginine biosynthesis via acetylated intermediates in microorganisms must be revised in light of recent enzymatic and genomic investigations. The enzyme N-acetylglutamate synthase (NAGS), which used to be considered responsible for the first committed step of the pathway, is present in a limited number of bacterial phyla only and is absent from Archaea. In many Bacteria, shorter proteins related to the Gcn5-related N-acetyltransferase family appear to acetylate l-glutamate; some are clearly similar to the C-terminal, acetyl-coenzyme A (CoA) binding domain of classical NAGS, while others are more distantly related. Short NAGSs can be single gene products, as in Mycobacterium spp. and Thermus spp., or fused to the enzyme catalyzing the last step of the pathway (argininosuccinase), as in members of the Alteromonas-Vibrio group. How these proteins bind glutamate remains to be determined. In some Bacteria, a bifunctional ornithine acetyltransferase (i.e., using both acetylornithine and acetyl-CoA as donors of the acetyl group) accounts for glutamate acetylation. In many Archaea, the enzyme responsible for glutamate acetylation remains elusive, but possible connections with a novel lysine biosynthetic pathway arose recently from genomic investigations. In some Proteobacteria (notably Xanthomonadaceae) and Bacteroidetes, the carbamoylation step of the pathway appears to involve N-acetylornithine or N-succinylornithine rather than ornithine. The product N-acetylcitrulline is deacetylated by an enzyme that is also involved in the provision of ornithine from acetylornithine; this is an important metabolic function, as ornithine itself can become essential as a source of other metabolites. This review insists on the biochemical and evolutionary implications of these findings.     

The late Maastrichtian nannofossil record of climate change in the South Atlantic DSDP Hole 525A

The phytoplankton response (calcareous nannofossils) to the Late Maastrichtian climate evolution is investigated in the South Atlantic DSDP Hole 525A and compared to published geochemical and micropaleontological data. The results point to a succession of dramatic climatic fluctuations. “Cool-water indicators” (Ahmuellerella octoradiata, Kamptnerius magnificus and Nephrolithus frequens) suggest cool surface water conditions prevailed during Chron C30n. At the top of C30n, their sudden drop in abundance, the last occurrence of B. constans and the concomitant increase in the tropical species Micula murus suggest warming and lower surface water productivity. An M. murus acme within Chron C29r reflects maximum warming. During the last 100 kyr of the Maastrichtian, the decrease in M. murus and increase in cool-water indicators reflect rapid cooling with the cool climate persisting over. The calcareous nannoplankton response to climate change correlate with similar findings in the Equatorial Atlantic Hole 1258A and parallels the stable isotope record of planktic and benthic foraminifera of DSDP Hole 525A as well as the decline in ¹⁸⁷Os/¹⁸⁸Os. Comparison of this marine record and the continental climate record in North America suggests a link between Deccan volcanism and the late Maastrichtian warm event.     

Characterization of pre-insertion loci of de novo L1 insertions

The human Long Interspersed Element-1 (LINE-1) and the Short Interspersed Element (SINE) Alu comprise 28% of the human genome. They share the same L1-encoded endonuclease for insertion, which recognizes an A+T-rich sequence. Under a simple model of insertion distribution, this nucleotide preference would lead to the prediction that the populations of both elements would be biased towards A+T-rich regions. Genomic L1 elements do show an A+T-rich bias. In contrast, Alu is biased towards G+C-rich regions when compared to the genome average. Several analyses have demonstrated that relatively recent insertions of both elements show less G+C content bias relative to older elements. We have analyzed the repetitive element and G+C composition of more than 100 pre-insertion loci derived from de novo L1 insertions in cultured human cancer cells, which should represent an evolutionarily unbiased set of insertions. An A+T-rich bias is observed in the 50 bp flanking the endonuclease target site, consistent with the known target site for the L1 endonuclease. The L1, Alu, and G+C content of 20 kb of the de novo pre-insertion loci shows a different set of biases than that observed for fixed L1s in the human genome. In contrast to the insertion sites of genomic L1s, the de novo L1 pre-insertion loci are relatively L1-poor, Alu-rich and G+C neutral. Finally, a statistically significant cluster of de novo L1 insertions was localized in the vicinity of the c-myc gene. These results suggest that the initial insertion preference of L1, while A+T-rich in the initial vicinity of the break site, can be influenced by the broader content of the flanking genomic region and have implications for understanding the dynamics of L1 and Alu distributions in the human genome.     

GeneBins: a database for classifying gene expression data,with application to plant genome arrays

Background  

To interpret microarray experiments, several ontological analysis tools have been developed. However, current tools are limited to specific organisms.     

The shunt from the cyclooxygenase to lipoxygenase pathway in human osteoarthritic subchondral osteoblasts is linked with a variable expression of the 5-lipoxygenase-activating protein

Osteoarthritis (OA) is characterized by articular cartilage degradation and hypertrophic bone changes with osteophyte formation and abnormal bone remodeling. Two groups of OA patients were identified via the production of variable and opposite levels of prostaglandin E₂ (PGE₂) or leukotriene B₄ (LTB₄) by subchondral osteoblasts, PGE₂ levels discriminating between low and high subgroups. We studied whether the expression of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) is responsible for the shunt from prostaglandins to leukotrienes. FLAP mRNA levels varied in low and high OA groups compared with normal, whereas mRNA levels of 5-LO were similar in all osteoblasts. Selective inhibition of cyclooxygenase-2 (COX-2) with NS-398-stimulated FLAP expression in the high OA osteoblasts subgroup, whereas it was without effect in the low OA osteoblasts subgroup. The addition of PGE₂ to the low OA osteoblasts subgroup decreased FLAP expression but failed to affect it in the high OA osteoblasts subgroup. LTB₄ levels in OA osteoblasts were stimulated about twofold by 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) plus transforming growth factor-β (TGF-β), a situation corresponding to their effect on FLAP mRNA levels. Treatments with 1,25(OH)₂D₃ and TGF-β also modulated PGE₂ production. TGF-β stimulated PGE₂ production in both OA osteoblast groups, whereas 1,25(OH)₂D₃ alone had a limited effect but decreased the effect of TGF-β in the low OA osteoblasts subgroup. This modulation of PGE₂ production was mirrored by the synthesis of COX-2. IL-18 levels were only slightly increased in a subgroup of OA osteoblasts compared with normal; however, no relationship was observed overall between IL-18 and PGE₂ levels in normal and OA osteoblasts. These results suggest that the shunt from the production of PGE₂ to LTB₄ is through regulation of the expression of FLAP, not 5-LO, in OA osteoblasts. The expression of FLAP in OA osteoblasts is also modulated differently by 1,25(OH)₂D₃ and TGF-β depending on their endogenous low and high PGE₂ levels.     

Alkyne-azide click chemistry mediated carbanucleosides synthesis

Hitherto unknown 1,4-disubstituted-[1,2,3]-triazolo-4',4'-dihydroxymethyl-3'-deoxy carbanucleosides were synthesized based on a "click approach." Various alkynes were introduced on a key azido intermediate by the "click" 1,3-dipolar Huisgen cycloaddition. Their antiviral activities and cellular toxicities were evaluated on vaccinia virus. None of the synthesized compounds exhibited a significant antiviral activity.     

Transition metal complexes bearing atropisomeric saturated NHC ligands

From achiral imidazolinium salts, chiral transition metal complexes containing an N-heterocyclic carbene (NHC) ligand were prepared (metal = palladium, copper, silver, gold, rhodium). Axial chirality in these complexes results from the formation of the metal-carbene bond leading to the restriction of rotation of dissymmetric N-aryl substituents about the C–N bond. When these complexes exhibited a sufficient configurational stability, a resolution by chiral high-performance liquid chromatography (HPLC) on preparative scale enabled isolation of enantiomers with excellent enantiopurities (>99% ee) and good yields. A study of the enantiomerization barriers revealed the effect of the backbone nature as well as the type of transition metal on its values. Nevertheless, the evaluation of palladium-based complexes in asymmetric intramolecular α-arylation of amides demonstrated that the ability to induce an enantioselectivity cannot be correlated to the configurational stability of the precatalysts.     

Induction of Photosynthetic Carbon Fixation in Anoxia Relies on Hydrogenase Activity and Proton-Gradient Regulation-Like1-Mediated Cyclic Electron Flow in Chlamydomonas reinhardtii

The model green microalga Chlamydomonas reinhardtii is frequently subject to periods of dark and anoxia in its natural environment. Here, by resorting to mutants defective in the maturation of the chloroplastic oxygen-sensitive hydrogenases or in Proton-Gradient Regulation-Like1 (PGRL1)-dependent cyclic electron flow around photosystem I (PSI-CEF), we demonstrate the sequential contribution of these alternative electron flows (AEFs) in the reactivation of photosynthetic carbon fixation during a shift from dark anoxia to light. At light onset, hydrogenase activity sustains a linear electron flow from photosystem II, which is followed by a transient PSI-CEF in the wild type. By promoting ATP synthesis without net generation of photosynthetic reductants, the two AEF are critical for restoration of the capacity for carbon dioxide fixation in the light. Our data also suggest that the decrease in hydrogen evolution with time of illumination might be due to competition for reduced ferredoxins between ferredoxin-NADP⁺ oxidoreductase and hydrogenases, rather than due to the sensitivity of hydrogenase activity to oxygen. Finally, the absence of the two alternative pathways in a double mutant pgrl1 hydrogenase maturation factor G-2 is detrimental for photosynthesis and growth and cannot be compensated by any other AEF or anoxic metabolic responses. This highlights the role of hydrogenase activity and PSI-CEF in the ecological success of microalgae in low-oxygen environments.Unicellular photosynthetic organisms such as the green alga Chlamydomonas reinhardtii frequently experience anoxic conditions in their natural habitat, especially during the night when the microbial community consumes the available oxygen. Under anoxia, lack of ATP synthesis by F₁F_O ATP synthase (EC 3.6.3.14) due to the absence of mitochondrial respiration is compensated by the activity of various plant- and bacterial-type fermentative enzymes that drive a sustained glycolytic activity (Mus et al., 2007; Terashima et al., 2010; Grossman et al., 2011; Yang et al., 2014). In C. reinhardtii, upstream glycolytic enzymes, including the reversible glyceraldehyde 3-P dehydrogenase, are located in the chloroplast (Johnson and Alric, 2012). This last enzyme is shared by the glycolysis (oxidative activity) and the Calvin-Benson-Bassham (CBB) cycle (reductive activity; Johnson and Alric, 2013). In dark anoxic conditions, the CBB cycle is inactive, thus avoiding wasteful using up of available ATP and depletion of the required intermediates for glycolysis. On the other side, ability of microalgae to perform photosynthetic carbon fixation when transferred from dark to light in the absence of oxygen might also be critical for adaptation to their environment. In such conditions, not only the linear electron flow (LEF) to Rubisco, but also alternative electron flow (AEF) toward oxygen (chlororespiration, Mehler reaction, and mitochondrial respiration; for review, see Miyake, 2010; Peltier et al., 2010; Cardol et al., 2011) is impaired. Thus, cells need to circumvent a paradoxical situation: the activity of the CBB cycle requires the restoration of the cellular ATP, but the chloroplastic F₁F_O ATP synthase activity is compromised by the impairment of most of the photosynthetic electron flows that usually generate the proton motive force in oxic conditions. Other AEFs, specific to anoxic conditions, should therefore be involved to promote ATP synthesis without net synthesis of NADPH and explain the light-induced restoration of CBB cycle activity.Among enzymes expressed in anoxia, the oxygen-sensitive hydrogenases (HYDA1 and HYDA2 in C. reinhardtii) catalyze the reversible reduction of protons into molecular hydrogen from the oxidation of reduced ferredoxins (FDXs; Florin et al., 2001). Although hydrogen metabolism in microalgae has been largely studied in the last 15 years in perspective of promising future renewable energy carriers (Melis et al., 2000; Kruse et al., 2005; Ghirardi et al., 2009), the physiological role of such an oxygen-sensitive enzyme linked to the photosynthetic pathway has been poorly considered. The 40-year-old proposal that H₂ evolution by hydrogenase is involved in induction of photosynthetic electron transfer after anoxic incubation (Kessler, 1973; Schreiber and Vidaver, 1974) has been only recently demonstrated in C. reinhardtii. Gas exchange measurements showed that H₂ evolution occurs prior to CO₂ fixation upon illumination (Cournac et al., 2002). At light onset after a prolonged period in dark anoxic conditions, the photosynthetic electron flow is mainly a LEF toward hydrogenase (Godaux et al., 2013), and lack of hydrogenase activity in hydrogenase maturation factor EF (hydEF) mutant strain deficient in hydrogenases maturation (Posewitz et al., 2004) induces a lag in induction of PSII activity (Ghysels et al., 2013). In cyanobacteria, the bidirectional Ni-Fe hydrogenase might also work as an electron valve for disposal of electrons generated at the onset of illumination of cells (Cournac et al., 2004) or when excess electrons are generated during photosynthesis, preventing the slowing of the electron transport chain under stress conditions (Appel et al., 2000; Carrieri et al., 2011). The bidirectional Ni-Fe hydrogenase could also dispose of excess of reducing equivalents during fermentation in dark anaerobic conditions, helping to generate ATP and maintaining homeostasis (Barz et al., 2010). A similar role for hydrogenase in setting the redox poise in the chloroplast of C. reinhardtii in anoxia has been recently uncovered (Clowez et al., 2015).Still, the physiological and evolutionary advantages of hydrogenase activity have not been demonstrated so far, and the mechanism responsible for the cessation of hydrogen evolution remains unclear. In this respect, at least three hypotheses have been formulated: (1) the inhibition of hydrogenase by O₂ produced by water photolysis (Ghirardi et al., 1997; Cohen et al., 2005), (2) the competition between ferredoxin-NADP⁺ oxidoreductase (FNR) and hydrogenase activity for reduced FDX (Yacoby et al., 2011), and (3) the inhibition of electron supply to hydrogenases by the proton gradient generated by another AEF, the cyclic electron flow around PSI (PSI-CEF; Tolleter et al., 2011). First described by Arnon (1955), PSI-CEF consists in a reinjection of electrons from reduced FDX or NADPH pool in the plastoquinone (PQ) pool. By generating an additional transthylakoidal proton gradient without producing reducing power, this AEF thus contributes to adjust the ATP/NADPH ratio for carbon fixation in various energetic unfavorable conditions including anoxia (Tolleter et al., 2011; Alric, 2014), high light (Tolleter et al., 2011; Johnson et al., 2014), or low CO₂ (Lucker and Kramer, 2013). In C. reinhardtii, two pathways have been suggested to be involved in PSI-CEF: (1) a type II NAD(P)H dehydrogenase (NDA2; Jans et al., 2008) driving the electrons from NAD(P)H to the PQ pool and (2) a pathway involving Proton Gradient Regulation (PGR) proteins where electrons from reduced FDXs return to the PQ pool or cytochrome b₆f. Not fully understood, this latter pathway comprises at least Proton Gradient Regulation5 (PGR5) and Proton-Gradient Regulation-Like1 (PGRL1) proteins (Iwai et al., 2010; Tolleter et al., 2011; Johnson et al., 2014) and is the major route for PSI-CEF in C. reinhardtii cells placed in anoxia (Alric, 2014).In this work, we took advantage of specific C. reinhardtii mutants defective in hydrogenase activity and PSI-CEF to study photosynthetic electron transfer after a period of dark anoxic conditions. Based on biophysical and physiological complementary studies, we demonstrate that at least hydrogenase activity or PSI-CEF is compulsory for the activity of the CBB cycle and for the survival of the cells submitted to anoxic conditions in their natural habitat.