RESUMEN
Plants thrive in diverse environments, where root-microbe interactions play a pivotal role. Date palm (Phoenix dactylifera L.), with its genetic diversity and resilience, is an ideal model for studying microbial adaptation to different genotypes and stresses. This study aimed to analyze the bacterial and fungal communities associated with traditional date palm cultivars and the widely cultivated "Deglet Nour" were explored using metabarcoding approaches. The microbial diversity analysis identified a rich community with 13,189 bacterial and 6442 fungal Amplicon Sequence Variants (ASVs). Actinobacteriota, Proteobacteria, and Bacteroidota dominated bacterial communities, while Ascomycota dominated fungal communities. Analysis of the microbial community revealed the emergence of two distinct clusters correlating with specific date palm cultivars, but fungal communities showed higher sensitivity to date palm genotype variations compared to bacterial communities. The commercial cultivar "Deglet Nour" exhibited a unique microbial composition enriched in pathogenic fungal taxa, which was correlated with its genetic distance. Overall, our study contributes to understanding the complex interactions between date palm genotypes and soil microbiota, highlighting the genotype role in microbial community structure, particularly among fungi. These findings suggest correlations between date palm genotype, stress tolerance, and microbial assembly, with implications for plant health and resilience. Further research is needed to elucidate genotype-specific microbial interactions and their role in enhancing plant resilience to environmental stresses.
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Bacterias , Hongos , Microbiota , Phoeniceae , Microbiología del Suelo , Phoeniceae/microbiología , Phoeniceae/genética , Bacterias/clasificación , Bacterias/genética , Bacterias/aislamiento & purificación , Hongos/genética , Hongos/clasificación , Hongos/aislamiento & purificación , Hongos/fisiología , Genotipo , Raíces de Plantas/microbiología , Suelo/químicaRESUMEN
Iron insertion into porphyrins is an essential step in heme biosynthesis. In the coproporphyrin-dependent pathway, specific to monoderm bacteria, this reaction is catalyzed by the monomeric enzyme coproporphyrin ferrochelatase. In addition to the mechanistic details of the metalation of the porphyrin, the identification of the substrate access channel for ferrous iron to the active site is important to fully understand this enzymatic system. In fact, whether the iron reaches the active site from the distal or the proximal porphyrin side is still under debate. In this study we have thoroughly addressed this question in Listeria monocytogenes coproporphyrin ferrochelatase by X-ray crystallography, steady-state and pre-steady-state imidazole ligand binding studies, together with a detailed spectroscopic characterization using resonance Raman and UV-vis absorption spectroscopies in solution. Analysis of the X-ray structures of coproporphyrin ferrochelatase-coproporphyrin III crystals soaked with ferrous iron shows that iron is present on both sides of the porphyrin. The kinetic and spectroscopic study of imidazole binding to coproporphyrin ferrochelataseiron coproporphyrin III clearly indicates the presence of two possible binding sites in this monomeric enzyme that influence each other, which is confirmed by the observed cooperativity at steady-state and a biphasic behavior in the pre-steady-state experiments. The current results are discussed in the context of the entire heme biosynthetic pathway and pave the way for future studies focusing on protein-protein interactions.
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OBJECTIVES: The sweet chestnut Castanea sativa Mill. is the only native Castanea species in Europe, and it is a tree of high economic value that provides appreciated fruits and valuable wood. In this study, we assembled a high-quality nuclear genome of the ancient Italian chestnut variety 'Marrone di Chiusa Pesio' using a combination of Oxford Nanopore Technologies long reads, whole-genome and Omni-C Illumina short reads. DATA DESCRIPTION: The genome was assembled into 238 scaffolds with an N50 size of 21.8 Mb and an N80 size of 7.1 Mb for a total assembled sequence of 750 Mb. The BUSCO assessment revealed that 98.6% of the genome matched the embryophyte dataset, highlighting good completeness of the genetic space. After chromosome-level scaffolding, 12 chromosomes with a total length of 715.8 and 713.0 Mb were constructed for haplotype 1 and haplotype 2, respectively. The repetitive elements represented 37.3% and 37.4% of the total assembled genome in haplotype 1 and haplotype 2, respectively. A total of 57,653 and 58,146 genes were predicted in the two haplotypes, and approximately 73% of the genes were functionally annotated using the EggNOG-mapper. The assembled genome will be a valuable resource and reference for future chestnut breeding and genetic improvement.
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Cromosomas de las Plantas , Fagaceae , Genoma de Planta , Fagaceae/genética , Genoma de Planta/genética , Cromosomas de las Plantas/genética , Haplotipos/genética , Anotación de Secuencia MolecularRESUMEN
Ferrochelatases catalyze the insertion of ferrous iron into the porphyrin during the heme b biosynthesis pathway, which is fundamental for both prokaryotes and eukaryotes. Interestingly, in the active site of ferrochelatases, the proximal ligand coordinating the porphyrin iron of the product is not conserved, and its catalytic role is still unclear. Here we compare the L. monocytogenes bacterial coproporphyrin ferrochelatase native enzyme together with selected variants, where the proximal Tyr residue was replaced by a His (i.e. the most common ligand in heme proteins), a Met or a Phe (as in human and actinobacterial ferrochelatases, respectively), in their Fe(III), Fe(II) and Fe(II)-CO adduct forms. The study of the active site structure and the activity of the proteins in solution has been performed by UV-vis electronic absorption and resonance Raman spectroscopies, biochemical characterization, and classical MD simulations. All the mutations alter the H-bond interactions between the iron porphyrin propionate groups and the protein, and induce effects on the activity, depending on the polarity of the proximal ligand. The overall results confirm that the weak or non-existing coordination of the porphyrin iron by the proximal residue is essential for the binding of the substrate and the release of the final product.
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Ferroquelatasa , Porfirinas , Humanos , Dominio Catalítico , Ferroquelatasa/química , Ferroquelatasa/metabolismo , Compuestos Férricos , Ligandos , Porfirinas/química , Hierro/química , Compuestos Ferrosos/metabolismoRESUMEN
The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria in 2015 by Dailey et al. triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here, we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely, His182Ala (H182A) and Glu263Gln (E263Q), involving two key active site residues. Interestingly, both variants are active only toward the physiological substrate coproporphyrin III but inactive toward protoporphyrin IX. In addition, E263 exchange impairs the final oxidation step from ferrous coproheme to ferric coproheme. The characteristics of the active site in the context of the residues involved and the substrate binding properties are discussed here using structural and functional means, providing a further contribution to the deciphering of this enigmatic reaction mechanism.
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Dominio Catalítico , Coproporfirinas , Ferroquelatasa , Ácido Glutámico , Histidina , Protoporfirinas , Ferroquelatasa/metabolismo , Ferroquelatasa/química , Ferroquelatasa/genética , Coproporfirinas/metabolismo , Coproporfirinas/química , Protoporfirinas/metabolismo , Protoporfirinas/química , Histidina/metabolismo , Histidina/química , Histidina/genética , Ácido Glutámico/metabolismo , Ácido Glutámico/química , Ácido Glutámico/genética , Hemo/metabolismo , Hemo/química , Especificidad por Sustrato , Modelos Moleculares , Oxidación-Reducción , Cinética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , CatálisisRESUMEN
Most hemoproteins display an all-α-helical fold, showing the classical three on three (3/3) globin structural arrangement characterized by seven or eight α-helical segments that form a sandwich around the heme. Over the last decade, a completely distinct class of heme-proteins called nitrobindins (Nbs), which display an all-ß-barrel fold, has been identified and characterized from both structural and functional perspectives. Nbs are ten-stranded anti-parallel all-ß-barrel heme-proteins found across the evolutionary ladder, from bacteria to Homo sapiens. Myoglobin (Mb), commonly regarded as the prototype of monomeric all-α-helical globins, is involved along with the oligomeric hemoglobin (Hb) in diatomic gas transport, storage, and sensing, as well as in the detoxification of reactive nitrogen and oxygen species. On the other hand, the function(s) of Nbs is still obscure, even though it has been postulated that they might participate to O2/NO signaling and metabolism. This function might be of the utmost importance in poorly oxygenated tissues, such as the eye's retina, where a delicate balance between oxygenation and blood flow (regulated by NO) is crucial. Dysfunction in this balance is associated with several pathological conditions, such as glaucoma and diabetic retinopathy. Here a detailed comparison of the structural, spectroscopic, and functional properties of Mb and Nbs is reported to shed light on the similarities and differences between all-α-helical and all-ß-barrel heme-proteins.
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Globinas , Mioglobina , Humanos , Globinas/química , Hemo/química , Hemoglobinas/química , Mioglobina/química , Análisis EspectralRESUMEN
BACKGROUND: Date palm (Phoenix dactylifera L.) is the most widespread crop in arid and semi-arid regions and has great traditional and socioeconomic importance, with its fruit well-known for its high nutritional and health value. However, the genetic variation of date palm cultivars is often neglected. The advent of high-throughput sequencing has made possible the resequencing of whole organelle (mitochondria and chloroplast) genomes to explore the genetic diversity and phylogenetic relationships of cultivated plants with unprecedented detail. RESULTS: Whole organelle genomes of 171 Tunisian accessions (135 females and 36 males) were sequenced. Targeted bioinformatics pipelines were used to identify date palm haplotypes and genome variants, aiming to provide variant annotation and investigate patterns of evolutionary relationship. Our results revealed the existence of unique haplotypes, identified by 45 chloroplastic and 156 mitochondrial SNPs. Estimation of the effect of these SNPs on genes functions was predicted in silico. CONCLUSIONS: The results of this study have important implications, in the light of ongoing environmental changes, for the conservation and sustainable use of the genetic resources of date palm cultivars in Tunisia, where monoculture threatens biodiversity leading to genetic erosion. These data will be useful for breeding and genetic improvement programs of the date palm through selective cross-breeding.
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Genoma del Cloroplasto , Phoeniceae , Phoeniceae/genética , Filogenia , Fitomejoramiento , Cloroplastos/genética , Mitocondrias/genéticaRESUMEN
Understanding the reaction mechanism of enzymes at the molecular level is generally a difficult task, since many parameters affect the turnover. Often, due to high reactivity and formation of transient species or intermediates, detailed information on enzymatic catalysis is obtained by means of model substrates. Whenever possible, it is essential to confirm a reaction mechanism based on substrate analogues or model systems by using the physiological substrates. Here we disclose the ferrous iron incorporation mechanism, in solution, and in crystallo, by the coproporphyrin III-coproporphyrin ferrochelatase complex from the firmicute, pathogen, and antibiotic resistant, Listeria monocytogenes. Coproporphyrin ferrochelatase plays an important physiological role as the metalation represents the penultimate reaction step in the prokaryotic coproporphyrin-dependent heme biosynthetic pathway, yielding coproheme (ferric coproporphyrin III). By following the metal titration with resonance Raman spectroscopy and x-ray crystallography, we prove that upon metalation the saddling distortion becomes predominant both in the crystal and in solution. This is a consequence of the readjustment of hydrogen bond interactions of the propionates with the protein scaffold during the enzymatic catalysis. Once the propionates have established the interactions typical of the coproheme complex, the distortion slowly decreases, to reach the almost planar final product.
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Coproporfirinas , Hierro , Coproporfirinas/metabolismo , Hierro/metabolismo , Ferroquelatasa/química , Ferroquelatasa/metabolismo , Propionatos/química , CatálisisRESUMEN
The European beech (Fagus sylvatica L.) is one of the most widespread forest trees in Europe whose distribution and intraspecific diversity has been largely shaped by repeated glacial cycles. Previous studies, mainly based on palaeobotanical evidence and a limited set of chloroplast and nuclear genetic markers, highlighted a complex phylogeographic scenario, with southern and western Europe characterized by a rather heterogeneous genetic structure, as a result of recolonization from different glacial refugia. Despite its ecological and economic importance, the genome of this broad-leaved tree has only recently been assembled, and its intra-species genomic diversity is still largely unexplored. Here, we performed whole-genome resequencing of nine Italian beech individuals sampled from two stands located in the Alpine and Apennine mountain ranges. We investigated patterns of genetic diversity at chloroplast, mitochondrial and nuclear genomes and we used chloroplast genomes to reconstruct a temporally-resolved phylogeny. Results allowed us to test European beech differentiation on a whole-genome level and to accurately date their divergence time. Our results showed comparable, relatively high levels of genomic diversity in the two populations and highlighted a clear differentiation at chloroplast, mitochondrial and nuclear genomes. The molecular clock analysis indicated an ancient split between the Alpine and Apennine populations, occurred between the Günz and the Riss glaciations (approximately 660 kyrs ago), suggesting a long history of separation for the two gene pools. This information has important conservation implications in the context of adaptation to ongoing climate changes.
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Fagus , Humanos , Fagus/genética , Europa (Continente) , Italia , Filogeografía , Filogenia , ÁrbolesRESUMEN
This work focuses on the carbon monoxide adducts of the wild-type and selected variants of the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae complexed with coproheme, monovinyl monopropionyl deuteroheme (MMD), and heme b. The UV - vis and resonance Raman spectroscopies together with the molecular dynamics simulations clearly show that the wild-type coproheme-CO adduct is characterized by two CO conformers, one hydrogen-bonded to the distal H118 residue and the other showing a weak polar interaction with the distal cavity. Instead, upon conversion to heme b, i.e. after decarboxylation of propionates 2 and 4 and rotation by 90o of the porphyrin ring inside the cavity, CO probes a less polar environment. In the absence of the H118 residue, both coproheme and heme b complexes form only the non-H-bonded CO species. The unrotated MMD-CO adduct as observed in the H118F variant, confirms that decarboxylation of propionate 2 only, does not affect the heme cavity. The rupture of both the H-bonds involving propionates 2 and 4 destabilizes the porphyrin inside the cavity with the subsequent formation of a CO adduct in an open conformation. In addition, in this work we present data on CO binding to reversed heme b, obtained by hemin reconstitution of the H118A variant, and to heme d, obtained by addition of an excess of hydrogen peroxide. The results will be discussed and compared with those reported for the representatives of the firmicute clade.
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Carboxiliasas , Corynebacterium diphtheriae , Monóxido de Carbono/metabolismo , Propionatos/química , Hemo/química , Espectrometría Raman , Carboxiliasas/químicaRESUMEN
Monoderm bacteria accumulate heme b via the coproporphyrin-dependent biosynthesis pathway. In the final step, in the presence of two molecules of H2O2, the propionate groups of coproheme at positions 2 and 4 are decarboxylated to form vinyl groups by coproheme decarboxylase (ChdC), in a stepwise process. Decarboxylation of propionate 2 produces an intermediate that rotates by 90° inside the protein pocket, bringing propionate 4 near the catalytic tyrosine, to allow the second decarboxylation step. The active site of ChdCs is stabilized by an extensive H-bond network involving water molecules, specific amino acid residues, and the propionate groups of the porphyrin. To evaluate the role of these H-bonds in the pocket stability and enzyme functionality, we characterized, via resonance Raman and electronic absorption spectroscopies, single and double mutants of the actinobacterial pathogen Corynebacterium diphtheriae ChdC complexed with coproheme and heme b. The selective elimination of the H-bond interactions between propionates 2, 4, 6, and 7 and the polar residues of the pocket allowed us to establish the role of each H-bond in the catalytic reaction and to follow the changes in the interactions from the substrate to the product.
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Carboxiliasas , Corynebacterium diphtheriae , Hemo/metabolismo , Enlace de Hidrógeno , Propionatos/química , Peróxido de Hidrógeno/química , Corynebacterium diphtheriae/metabolismo , Carboxiliasas/químicaRESUMEN
Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV-vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies.
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Porfirinas , Porfirinas/química , Coproporfirinas/metabolismo , Propionatos , Dominio Catalítico , Ferroquelatasa/genética , Ferroquelatasa/química , Ferroquelatasa/metabolismo , Sitios de Unión , Hierro/metabolismoRESUMEN
The properties of water at interfaces have long been known to differ from those of bulk water in many distinctive ways. More recently, specific confinement effects different from mere interfacial effects have been discovered upon enclosing water in very narrow cylindrical pores and planar surfaces as offered by nanotubes and slit pores, respectively. Using experimental and theoretical THz spectroscopy, we elucidate nanoconfinement effects on the H-bond network of stratified water lamellae that are hosted within graphene-based two-dimensional pores. Characteristic confinement-induced changes of the THz response are traced back to the level of structural dynamics, notably distinct resonances due to intralayer and interlayer H-bonds at correspondingly low and high intermolecular stretching frequencies and impact of dangling (free) OH bonds at the water-graphene interface that enormously broaden the librational band in sufficiently narrow pores. The interplay of these molecular effects causes characteristic changes of the THz lineshape upon nanoconfining water.
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Nitrobindins (Nbs) are all-ß-barrel heme-proteins present in all the living kingdoms. Nbs inactivate reactive nitrogen species by sequestering NO, converting NO to HNO2, and isomerizing peroxynitrite to NO3- and NO2-. Here, the spectroscopic characterization of ferric Danio rerio Nb (Dr-Nb(III)) and NO scavenging through the reductive nitrosylation of the metal center are reported, both processes being relevant for the regulation of blood flow in fishes through poorly oxygenated tissues, such as retina. Both UV-Vis and resonance Raman spectroscopies indicate that Dr-Nb(III) is a mixture of a six-coordinated aquo- and a five-coordinated species, whose relative abundancies depend on pH. At pH ≤ 7.0, Dr-Nb(III) binds reversibly NO, whereas at pH ≥ 7.8 NO induces the conversion of Dr-Nb(III) to Dr-Nb(II)-NO. The conversion of Dr-Nb(III) to Dr-Nb(II)-NO is a monophasic process, suggesting that the formation of the transient Dr-Nb(III)-NO species is lost in the mixing time of the rapid-mixing stopped-flow apparatus (â¼ 1.5 ms). The pseudo-first-order rate constant for the reductive nitrosylation of Dr-Nb(III) is not linearly dependent on the NO concentration but tends to level off. Values of the rate-limiting constant (i.e., klim) increase linearly with the OH- concentration, indicating that the conversion of Dr-Nb(III) to Dr-Nb(II)-NO is limited by the OH--based catalysis. From the dependence of klim on [OH-], the value of the second-order rate constant kOH- was obtained (5.2 × 103 M-1 s-1). Reductive nitrosylation of Dr-Nb(III) leads to the inactivation of two NO molecules: one being converted to HNO2, and the other being tightly bound to the heme-Fe(II) atom.
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Hemo , Pez Cebra , Animales , Oxidación-Reducción , Cinética , Hemo/química , Hierro , Termodinámica , Compuestos Férricos/químicaRESUMEN
Nitrobindins (Nbs) are all-ß-barrel heme-proteins present in prokaryotes and eukaryotes. Although the physiological role(s) of Nbs are still unclear, it has been postulated that they are involved in the NO/O2 metabolism, which is particularly relevant in fishes for the oxygen supply. Here, the reactivity of ferrous Danio rerio Nb (Dr-Nb(II)) towards NO has been investigated from the spectroscopic and kinetic viewpoints and compared with those of Mycobacterium tuberculosis Nb, Arabidopsis thaliana Nb, Homo sapiens Nb, and Equus ferus caballus myoglobin. Between pHâ¯5.5 and 9.1 at 22.0⯰C, Dr-Nb(II) nitrosylation is a monophasic process; values of the second-order rate constant for Dr-Nb(II) nitrosylation and of the first-order rate constant for Dr-Nb(II)-NO denitrosylation are pH-independent ranging between 1.6â¯×â¯106â¯M-1â¯s-1 and 2.3â¯×â¯106â¯M-1â¯s-1 and between 5.3â¯×â¯10-2â¯s-1 and 8.2â¯×â¯10-2â¯s-1, respectively. Interestingly, both UV-Vis and EPR spectroscopies indicate that the heme-Fe(II) atom of Dr-Nb(II)-NO is five-coordinated. Kinetics of Dr-Nb(II) nitrosylation may reflect the ligand accessibility to the metal center, which is likely impaired by the crowded network of water molecules which shields the heme pocket from the bulk solvent. On the other hand, kinetics of Dr-Nb(II)-NO denitrosylation may reflect an easy pathway for the ligand escape into the outer solvent.
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Mioglobina , Pez Cebra , Animales , Compuestos Ferrosos/química , Hemo/química , Caballos , Cinética , Ligandos , Mioglobina/metabolismo , Oxígeno/química , Solventes , AguaRESUMEN
The actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae catalyzes the final reaction to generate heme b via the "coproporphyrin-dependent" heme biosynthesis pathway in the presence of hydrogen peroxide. The enzyme has a high reactivity toward H2O2 used for the catalytic reaction and in the presence of an excess of H2O2 new species are generated. Resonance Raman data, together with electronic absorption spectroscopy and mass spectrometry, indicate that an excess of hydrogen peroxide for both the substrate (coproheme) and product (heme b) complexes of this enzyme causes a porphyrin hydroxylation of ring C or D, which is compatible with the formation of an iron chlorin-type heme d species. A similar effect has been previously observed for other heme-containing proteins, but this is the first time that a similar mechanism is reported for a coproheme enzyme. The hydroxylation determines a symmetry lowering of the porphyrin macrocycle, which causes the activation of A2g modes upon Soret excitation with a significant change in their polarization ratios, the enhancement and splitting into two components of many Eu bands, and an intensity decrease of the non-totally symmetric modes B1g, which become polarized. This latter effect is clearly observed for the isolated ν10 mode upon either Soret or Q-band excitations. The distal His118 is shown to be an absolute requirement for the conversion to heme d. This residue also plays an important role in the oxidative decarboxylation, because it acts as a base for deprotonation and subsequent heterolytic cleavage of hydrogen peroxide.
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Arsenic is one of the most prevalent toxic elements in the environment, and its toxicity affects every organism. Arsenic resistance has mainly been observed in microorganisms, and, in bacteria, it has been associated with the presence of the Ars operon. In Saccharomyces cerevisiae, three genes confer arsenic resistance: ARR1, ARR2, and ARR3. Unlike bacteria, in which the presence of the Ars genes confers per se resistance to arsenic, most of the S. cerevisiae isolates present the three ARR genes, regardless of whether the strain is resistant or sensitive to arsenic. To assess the genetic features that make natural S. cerevisiae strains resistant to arsenic, we used a combination of comparative genomic hybridization, whole-genome sequencing, and transcriptomics profiling with microarray analyses. We observed that both the presence and the genomic location of multiple copies of the whole cluster of ARR genes were central to the escape from subtelomeric silencing and the acquisition of resistance to arsenic. As a result of the repositioning, the ARR genes were expressed even in the absence of arsenic. In addition to their relevance in improving our understanding of the mechanism of arsenic resistance in yeast, these results provide evidence for a new cluster of functionally related genes that are independently duplicated and translocated.
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Arsénico , Arsenitos , Arseniatos/toxicidad , Arsénico/toxicidad , Arsenitos/toxicidad , Hibridación Genómica Comparativa , Operón , Saccharomyces cerevisiae/genéticaRESUMEN
We produced a neuroglobin variant, namely, Ngb CDless, with the excised CDloop- and D-helix, directly joining the C- and E-helices. The CDless variant retained bis-His hexacoordination, and we investigated the role of the CDloop-D-helix unit in controlling the CO binding and structural dynamics by an integrative approach based on X-ray crystallography, rapid mixing, laser flash photolysis, resonance Raman spectroscopy, and molecular dynamics simulations. Rapid mixing and laser flash photolysis showed that ligand affinity was unchanged with respect to the wild-type protein, albeit with increased on and off constants for rate-limiting heme iron hexacoordination by the distal His64. Accordingly, resonance Raman spectroscopy highlighted a more open distal pocket in the CO complex that, in agreement with MD simulations, likely involves His64 swinging inward and outward of the distal heme pocket. Ngb CDless displays a more rigid overall structure with respect to the wild type, abolishing the structural dynamics of the CDloop-D-helix hypothesized to mediate its signaling role, and it retains ligand binding control by distal His64. In conclusion, this mutant may represent a tool to investigate the involvement of CDloop-D-helix in neuroprotective signaling in a cellular or animal model.
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Neuroglobina/química , Animales , Hemo/química , Ligandos , Ratones , Neuroglobina/metabolismoRESUMEN
Hydration free energies are dictated by a subtle balance of hydrophobic and hydrophilic interactions. We present here a spectroscopic approach, which gives direct access to the two main contributions: Using THz-spectroscopy to probe the frequency range of the intermolecular stretch (150-200â cm-1 ) and the hindered rotations (450-600â cm-1 ), the local contributions due to cavity formation and hydrophilic interactions can be traced back. We show that via THz calorimetry these fingerprints can be correlated 1 : 1 with the group specific solvation entropy and enthalpy. This allows to deduce separately the hydrophobic (i.e. cavity formation) and hydrophilic contributions to thermodynamics, as shown for hydrated alcohols as a case study. Accompanying molecular dynamics simulations quantitatively support our experimental results. In the future our approach will allow to dissect hydration contributions in inhomogeneous mixtures and under non-equilibrium conditions.
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Agua , Entropía , Interacciones Hidrofóbicas e Hidrofílicas , Soluciones , Análisis Espectral , Termodinámica , Agua/químicaRESUMEN
Riparian ecosystems, in long-time developed regions, are among the most heavily impacted by human activities; therefore, the distribution of tree riparian species, such as Ulmus laevis, is highly affected. This phenomenon is particularly relevant at the margins of the natural habitat of the species, where populations are small and rare. In these cases, it is difficult to distinguish between relics or introductions, but it is relevant for the restoration of natural habitats and conservation strategies. The aim of this study was to study the phylogeography of the southern distribution of the species. We sequenced the entire chloroplast (cp) genomes of 54 individuals from five sampled populations across different European regions to highlight polymorphisms and analyze their distribution. Thirty-two haplotypes were identified. All the sampled populations showed private haplotypes that can be considered an indicator of long-term residency, given the low mutation rate of organellar DNA. The network of all haplotypes showed a star-like topology, and Serbian haplotypes were present in all branches. The Balkan population showed the highest level of nucleotide and genetic diversity. Low genetic differentiation between populations was observed but we found a significant differentiation among Serbia vs. other provenances. Our estimates of divergent time of U. laevis samples highlight the early split of above all Serbian individuals from other populations, emphasizing the reservoir role of white elm genetic diversity of Serbian population.