Metabolic enzymes from psychrophilic bacteria: Challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi

The enzyme ornithine carbamoyltransferase (OTCase) of Motitella abyssi (OTCase(Mab)), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase(Mab) displays maximal activity at rather low temperatures (23 to 25degreesC) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase(Mab) displays higher K-m values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue delta-N-phosphonoacetyl-L-ornithine (PALO). OTCase(Mab) differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K-m values and the lower sensitivity to PALO. The K. for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k(cat)/K-m) of OTCases adapted to different temperatures showed that OTCase(Mab) activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K-m value for ornithine observed when the temperature is brought from 20 to 5degreesC. OTCase(Mab) adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits

Stability domains, substrate-induced conformational changes, and hinge-bending motions in a psychrophilic phosphoglycerate kinase: A microcalorimetric study

The cold-active phosphoglycerate kinase from the Antarctic bacterium Pseudomonas sp. TACII18 exhibits two distinct stability domains in the free, open conformation. It is shown that these stability domains do not match the structural N- and C-domains as the heat-stable domain corresponds to about 80 residues of the C-domain, including the nucleotide binding site, whereas the remaining of the protein contributes to the main heat-labile domain. This was demonstrated by spectroscopic and microcalorimetric analyses of the native enzyme, of its mutants, and of the isolated recombinant structural domains. It is proposed that the heat-stable domain provides a compact structure improving the binding affinity of the nucleotide, therefore increasing the catalytic efficiency at low temperatures. Upon substrate binding, the enzyme adopts a uniformly more stable closed conformation. Substrate-induced stability changes suggest that the free energy of ligand binding is converted into an increased conformational stability used to drive the hinge-bending motions and domain closure

Crystal structure of the cold-active aminopeptidase from Colwellia psychrerythraea, a close structural homologue of the human bifunctional leukotriene A4 hydrolase

peer reviewedThe crystal structure of a cold-active aminopeptidase (ColAP) from Colwellia psychrerythraea strain 34H has been determined, extending the number of crystal structures of the M1 metallopeptidase family to four among the 436 members currently identified. In agreement with their sequence similarity, the overall structure of ColAP displayed a high correspondence with leukotriene A4 hydrolase (LTA4H), a human bifunctional enzyme that converts leukotriene A4 (LTA4) in the potent chemoattractant leukotriene B4. Indeed, both enzymes are composed of three domains, an N-terminal saddle-like domain, a catalytic thermolysin-like domain, and a less conserved C-terminal alpha-helical flat spiral domain. Together, these domains form a deep cavity harboring the zinc binding site formed by residues included in the conserved HEXXHX(18)H motif. A detailed structural comparison of these enzymes revealed several plausible determinants of ColAP cold adaptation. The main differences involve specific amino acid substitutions, loop content and solvent exposure, complexity and distribution of ion pairs, and differential domain flexibilities. Such elements may act synergistically to allow conformational flexibility needed for an efficient catalysis in cold environments. Furthermore, the region of ColAP corresponding to the aminopeptidase active site of LTA4H is much more conserved than the suggested LTA4 substrate binding region. This observation supports the hypothesis that this region of the LTA4H active site has evolved in order to fit the lipidic substrate

Explorando el metagenoma del suelo antártico como fuente de nuevas enzimas adaptadas al frío y elementos génicos móviles

A partir de muestras de suelo antártico se obtuvo la metagenoteca PP1. Esta fue sometida a análisis funcionales y genotípicos para el aislamiento de nuevas enzimas adaptadas al frío con potenciales aplicaciones, y para la detección de elementos génicos asociados a la movilización de genes, respectivamente. Por tamizaje fenotípico se detectaron 14, 14, 3 y 11 clones productores de lipasas/esterasas, proteasas, amilasas y celulasas, respectivamente, con actividades máximas aparentes de 35 °C para las amilasas y lipasas, y de 35-55 °C para las celulasas, tal como se observó para otras enzimas adaptadas al frío. Sin embargo, una celulasa parece ser compatible con enzimas mesófilas, las que usualmente se mantienen activas hasta por sobre 60 °C. Este hecho probablemente esté asociado a un comportamiento psicrotolerante en los suelos antárticos. La metagenómica permite acceder a una nueva miríada de productos metabólicos con potenciales beneficios para aplicaciones biotecnológicas e industriales. Se detectaron los genes tipo intI y tnp por PCR, y sus productos génicos deducidos tuvieron identidades del 58 al 86 % y del 58 al 73 % con secuencias conocidas, respectivamente. Dos clones, BAC 27A-9 y BAC 14A-5, parecen presentar organizaciones sintéticas únicas, lo cual sugiere la existencia de rearreglos génicos probablemente debidos a divergencias evolutivas dentro del género o facilitados por la asociación de elementos de transposición. La evidencia de elementos génicos relacionados con el reclutamiento y la movilización de genes en ambientes extremos como la Antártida refuerza la hipótesis sobre el origen de algunos genes diseminados por elementos móviles entre los microorganismos asociados al ser humano.Metagenomic library PP1 was obtained from Antarctic soil samples. Both functional and genotypic metagenomic screening were used for the isolation of novel cold-adapted enzymes with potential applications, and for the detection of genetic elements associated with gene mobilization, respectively. Fourteen lipase/esterase-, 14 amylase-, 3 protease-, and 11 cellulase-producing clones were detected by activity-driven screening, with apparent maximum activities around 35 °C for both amylolytic and lipolytic enzymes, and 35-55 °C for cellulases, as observed for other cold-adapted enzymes. However, the behavior of at least one of the studied cellulases is more compatible to that observed for mesophilic enzymes. These enzymes are usually still active at temperatures above 60 °C, probably resulting in a psychrotolerant behavior in Antarctic soils. Metagenomics allows to access novel genes encoding for enzymatic and biophysic properties from almost every environment with potential benefits for biotechnological and industrial applications. Only intI- and tnp-like genes were detected by PCR, encoding for proteins with 58-86 %, and 58-73 % amino acid identity with known entries, respectively. Two clones, BAC 27A-9 and BAC 14A-5, seem to present unique syntenic organizations, suggesting the occurrence of gene rearrangements that were probably due to evolutionary divergences within the genus or facilitated by the association with transposable elements. The evidence for genetic elements related to recruitment and mobilization of genes (transposons/integrons) in an extreme environment like Antarctica reinforces the hypothesis of the origin of some of the genes disseminated by mobile elements among "human-associated" microorganisms.Fil: Berlemont, Renaud. Universite de Liege; BélgicaFil: Pipers, Delphine. Universite de Liege; BélgicaFil: Delsaute, Maud. Universite de Liege; BélgicaFil: Angiono, Federico. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; ArgentinaFil: Feller, Georges. Universite de Liege; BélgicaFil: Galleni, Moreno. Universite de Liege; BélgicaFil: Power, Pablo. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Microbiología, Inmunología y Biotecnología; Argentina. Universite de Liege; Bélgica. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentin

Fluctuations of linear statistics of half-heavy-tailed random matrices

publisher: Elsevier articletitle: Fluctuations of linear statistics of half-heavy-tailed random matrices journaltitle: Stochastic Processes and their Applications articlelink: http://dx.doi.org/10.1016/j.spa.2016.04.030 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved.publisher: Elsevier articletitle: Fluctuations of linear statistics of half-heavy-tailed random matrices journaltitle: Stochastic Processes and their Applications articlelink: http://dx.doi.org/10.1016/j.spa.2016.04.030 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved.publisher: Elsevier articletitle: Fluctuations of linear statistics of half-heavy-tailed random matrices journaltitle: Stochastic Processes and their Applications articlelink: http://dx.doi.org/10.1016/j.spa.2016.04.030 content_type: article copyright: © 2016 Elsevier B.V. All rights reserved

Chapitre 13. Séquestration du carbone et usage durable des savanes ouest-africaines : synergie ou antagonisme ?

Introduction Les écosystèmes de savane ouest-africaine couvrent de vastes superficies (5.106 km2), en grande majorité exploitées par l’agriculture et le pastoralisme (Mayaux et al., 2004). Ils associent des systèmes herbacés et arborés, les faciès de végétation étant fortement pilotés par la pluviosité annuelle selon un gradient latitudinal. Dans ces écosystèmes, pour des raisons principalement démographiques et techniques – et, demain, sans doute climatiques – les ressources carbonées se rar..

Decoding the Folding of Burkholderia glumae Lipase: Folding Intermediates En Route to Kinetic Stability

The lipase produced by Burkholderia glumae folds spontaneously into an inactive near-native state and requires a periplasmic chaperone to reach its final active and secretion-competent fold. The B. glumae lipase-specific foldase (Lif) is classified as a member of the steric-chaperone family of which the propeptides of α-lytic protease and subtilisin are the best known representatives. Steric chaperones play a key role in conferring kinetic stability to proteins. However, until present there was no solid experimental evidence that Lif-dependent lipases are kinetically trapped enzymes. By combining thermal denaturation studies with proteolytic resistance experiments and the description of distinct folding intermediates, we demonstrate that the native lipase has a kinetically stable conformation. We show that a newly discovered molten globule-like conformation has distinct properties that clearly differ from those of the near-native intermediate state. The folding fingerprint of Lif-dependent lipases is put in the context of the protease-prodomain system and the comparison reveals clear differences that render the lipase-Lif systems unique. Limited proteolysis unveils structural differences between the near-native intermediate and the native conformation and sets the stage to shed light onto the nature of the kinetic barrier

The Role of Response Elements Organization in Transcription Factor Selectivity: The IFN-β Enhanceosome Example

What is the mechanism through which transcription factors (TFs) assemble specifically along the enhancer DNA? The IFN-β enhanceosome provides a good model system: it is small; its components' crystal structures are available; and there are biochemical and cellular data. In the IFN-β enhanceosome, there are few protein-protein interactions even though consecutive DNA response elements (REs) overlap. Our molecular dynamics (MD) simulations on different motif combinations from the enhanceosome illustrate that cooperativity is achieved via unique organization of the REs: specific binding of one TF can enhance the binding of another TF to a neighboring RE and restrict others, through overlap of REs; the order of the REs can determine which complexes will form; and the alternation of consensus and non-consensus REs can regulate binding specificity by optimizing the interactions among partners. Our observations offer an explanation of how specificity and cooperativity can be attained despite the limited interactions between neighboring TFs on the enhancer DNA. To date, when addressing selective TF binding, attention has largely focused on RE sequences. Yet, the order of the REs on the DNA and the length of the spacers between them can be a key factor in specific combinatorial assembly of the TFs on the enhancer and thus in function. Our results emphasize cooperativity via RE binding sites organization