Life on Minerals: Binding Behaviors of Oligonucleotides on Zirconium Silicate and Its Inhibitory Activity for the Self-Cleavage of Hammerhead Ribozyme.

The role of minerals in the chemical evolution of RNA molecules is an important issue when considering the early stage of the Hadean Earth. In particular, the interaction between functional ribozymes and ancient minerals under simulated primitive conditions is a recent research focus. We are currently attempting to design a primitive RNA metabolic network which would function with minerals, and believe that the simulated chemical network of RNA molecules would be useful for evaluation of the chemical evolution from a simple RNA mixture to an RNA-based life-like system. First, we measured the binding interactions of oligonucleotides with four types of minerals; Aerosil silica, zirconium silicate, sepiolite, and montmorillonite. Oligonucleotides bound zirconium silicate and montmorillonite in the presence of MgCl2, and bound sepiolite both in the presence and absence of MgCl2, but they did not bind Aerosil. Based on the binding behavior, we attempted the self-cleavage reaction of the hammerhead ribozyme from an avocado viroid. This reaction was strongly inhibited by zirconium silicate, a compound regarded as mineral evidence for the existence of water. The present study suggests that the chemical evolution of functional RNA molecules requires specific conformational binding, resulting in efficient ribozyme function as well as zirconium silicate for the chemical evolution of biomolecules.

A High-Pressure, High-Temperature Flow Reactor Simulating the Hadean Earth Environment, with Application to the Pressure Dependence of the Cleavage of Avocado Viroid Hammerhead Ribozyme.

The RNA world hypothesis suggests that chemical networks consisting of functional RNA molecules could have constructed a primitive life-like system leading a first living system. The chemical evolution scenario of RNA molecules should be consistent with the Hadean Earth environment. We have demonstrated the importance of the environment at both high temperature and high pressure, using different types of hydrothermal flow reactor systems and high-pressure equipment. In the present study, we have attempted to develop an alternative easy-to-implement method for high-pressure measurements and demonstrate that the system is applicable as an efficient research tool for high-pressure experiments at pressures up to 30 MPa. We demonstrate the usefulness of the system by detecting the high-pressure influence for the self-cleavage of avocado hammerhead ribozyme (ASBVd(-):HHR) at 45-65 °C. A kinetic analysis of the high-pressure behavior of ASBVd(-):HHR shows that the ribozyme is active at 30 MPa and its activity is sensitive to pressures between 0.1-30 MPa. The surprising finding that such a short ribozyme is effective for self-cleavage at a high pressure suggests the importance of pressure as a factor for selection of adaptable RNA molecules towards an RNA-based life-like system in the Hadean Earth environment deep in the ocean.

Kinetic Study of the Avocado Sunblotch Viroid Self-Cleavage Reaction Reveals Compensatory Effects between High-Pressure and High-Temperature: Implications for Origins of Life on Earth.

A high pressure apparatus allowing one to study enzyme kinetics under pressure was used to study the self-cleavage activity of the avocado sunblotch viroid. The kinetics of this reaction were determined under pressure over a range up to 300 MPa (1-3000 bar). It appears that the initial rate of this reaction decreases when pressure increases, revealing a positive ΔV≠ of activation, which correlates with the domain closure accompanying the reaction and the decrease of the surface of the viroid exposed to the solvent. Although, as expected, temperature increases the rate of the reaction whose energy of activation was determined, it appeared that it does not significantly influence the ΔV≠ of activation and that pressure does not influence the energy of activation. These results provide information about the structural aspects or this self-cleavage reaction, which is involved in the process of maturation of this viroid. The behavior of ASBVd results from the involvement of the hammerhead ribozyme present at its catalytic domain, indeed a structural motif is very widespread in the ancient and current RNA world.

Ribozyme Chemistry: To Be or Not To Be under High Pressure.

The use of high hydrostatic pressure to investigate structure-function relationships in biomacromolecules in solution provides precise information about conformational changes and variations of the interactions between these macromolecules and the solvent, as well as the volume changes associated with their activity. The complementary use of osmotic pressure reveals quantitatively the extent and direction of the water exchanges between the macromolecules and the solvent and the number of water molecules involved in these exchanges. In this review, the chemistry of ribozymes and the influence of pressure is described. In the case of the hairpin ribozyme, pressure slowed down the self-cleavage reaction on the basis that the formation of the transition state involves a positive ΔV⧧ of activation and the release of 78 ± 4 water molecules. The self-cleaving activity of the hammerhead ribozyme is also slowed down by pressure on the basis of kinetic parameters and ΔVs comparable to those of the hairpin ribozymes. However, it appears that the solution of the hammerhead ribozyme used in this study contains two populations of molecules which differ by the values of these parameters. The results obtained in the case of small self-cleaving ribozymes containing adenine bulges are consistent with the hypothesis that these small RNAs that bind amino acids or peptides could have appeared in prebiotic chemistry under extreme conditions in deep-sea vents or hydrothermal surface sites.

Pressure-induced activation of latent dihydroorotase from Aquifex aeolicus as revealed by high pressure protein crystallography.

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated. It was previously shown that high pressure fully restores the activity of this isolated DHOase. On the basis of kinetic studies, site-directed mutagenesis and the use of peptides mimicking loop A, a loop that appears to block access to the active site, was proposed that this pressure-induced reactivation was due to the decrease in the volume of the system, -ΔV, resulting from the disruption of known ionic interactions between the loop and the main part of the protein. In this study, this interpretation is more precisely demonstrated by the determination of the crystallographic structure of isolated DHOase under pressure. In addition to the loop displacements, pressure induces a discrete rearrangement of the catalytic site aspartate 305, an effect that might additionally contribute to the reactivation of this enzyme.

Structural Insight into the Core of CAD.

The CAD complex catalyzes the first four reactions of the pyrimidine biosynthetic pathway. CAD exhibits allosteric regulaton, both negative and positive, covalent regulation by phosphorylation, and metabolite channeling. In this issue of Structure, Moreno-Morcillo et al. (2017) show that the dihydroorotase domain plays a crucial role in the establishment of the quaternary structure of this complex.

Activation of Latent Dihydroorotase from Aquifex aeolicus by Pressure.

Elevated hydrostatic pressure was used to probe conformational changes of Aquifex aeolicus dihydroorotase (DHO), which catalyzes the third step in de novo pyrimidine biosynthesis. The isolated protein, a 45-kDa monomer, lacks catalytic activity but becomes active upon formation of a dodecameric complex with aspartate transcarbamoylase (ATC). X-ray crystallographic studies of the isolated DHO and of the complex showed that association induces several major conformational changes in the DHO structure. In the isolated DHO, a flexible loop occludes the active site blocking the access of substrates. The loop is mostly disordered but is tethered to the active site region by several electrostatic and hydrogen bonds. This loop becomes ordered and is displaced from the active site upon formation of DHO-ATC complex. The application of pressure to the complex causes its time-dependent dissociation and the loss of both DHO and ATC activities. Pressure induced irreversible dissociation of the obligate ATC trimer, and as a consequence the DHO is also inactivated. However, moderate hydrostatic pressure applied to the isolated DHO subunit mimics the complex formation and reversibly activates the isolated subunit in the absence of ATC, suggesting that the loop has been displaced from the active site. This effect of pressure is explained by the negative volume change associated with the disruption of ionic interactions and exposure of ionized amino acids to the solvent (electrostriction). The interpretation that the loop is relocated by pressure was validated by site-directed mutagenesis and by inhibition by small peptides that mimic the loop residues.

The M1 family of vertebrate aminopeptidases: role of evolutionarily conserved tyrosines in the enzymatic mechanism of aminopeptidase B.

Aminopeptidase B (Ap-B), a member of the M1 family of Zn(2+)-aminopeptidases, removes basic residues at the NH2-terminus of peptides and is involved in the in vivo proteolytic processing of miniglucagon and cholecystokinin-8. M1 enzymes hydrolyze numerous different peptides and are implicated in many physiological functions. As these enzymes have similar catalytic mechanisms, their respective substrate specificity and/or catalytic efficiency must be based on subtle structural differences at or near the catalytic site. This leads to the hypothesis that each primary structure contains a consensus structural template, strictly necessary for aminopeptidase activity, and a specific amino acid environment localized in or outside the catalytic pocket that finely tunes the substrate specificity and catalytic efficiency of each enzyme. A multiple sequence alignment of M1 peptidases from vertebrates allowed to identify conserved tyrosine amino acids, which are members of this catalytic backbone. In the present work, site-directed mutagenesis and 3D molecular modeling of Ap-B were used to specify the role of four fully (Y281, Y229, Y414, and Y441) and one partially (Y409) conserved residues. Tyrosine to phenylalanine mutations allowed confirming the influence of the hydroxyl groups on the enzyme activity. These groups are implicated in the reaction mechanism (Y414), in substrate specificity and/or catalytic efficiency (Y409), in stabilization of essential amino acids of the active site (Y229, Y409) and potentially in the maintenance of its structural integrity (Y281, Y441). The importance of hydrogen bonds is verified by the Y229H substitution, which preserves the enzyme activity. These data provide new insights into the catalytic mechanism of Ap-B in the M1 family of aminopeptidases.

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid.

BACKGROUND: Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication. METHODS: In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors. RESULTS: Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with Ki of 17±5µM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with Ki of 32±5µM and IC50 of 177±5nM, respectively. CONCLUSIONS: This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups. GENERAL SIGNIFICANCE: This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.

Behavior of a hammerhead ribozyme in aqueous solution at medium to high temperatures.

The "RNA world" hypothesis proposes that--early in the evolution of life--RNA molecules played important roles both in information storage and in enzymatic functions. However, this hypothesis seems to be inconsistent with the concept that life may have emerged under hydrothermal conditions since RNA molecules are considered to be labile under such extreme conditions. Presently, the possibility that the last common ancestor of the present organisms was a hyperthermophilic organism which is important to support the hypothesis of the hydrothermal origin of life has been subject of strong discussions. Consequently, it is of importance to study the behavior of RNA molecules under hydrothermal conditions from the viewpoints of stability, catalytic functions, and storage of genetic information of RNA molecules and determination of the upper limit of temperature where life could have emerged. In the present work, self-cleavage of a natural hammerhead ribozyme was examined at temperatures 10-200 °C. Self-cleavage was investigated in the presence of Mg(2+), which facilitates and accelerates this reaction. Self-cleavage of the hammerhead ribozyme was clearly observed at temperatures up to 60 °C, but at higher temperatures self-cleavage occurs together with hydrolysis and with increasing temperature hydrolysis becomes dominant. The influence of the amount of Mg(2+) on the reaction rate was also investigated. In addition, we discovered that the reaction proceeds in the presence of high concentrations of monovalent cations (Na(+) or K(+)), although very slowly. Furthermore, at high temperatures (above 60 °C), monovalent cations protect the ribozyme against degradation.

High-pressure analysis of a hammerhead ribozyme from Chrysanthemum chlorotic mottle viroid reveals two different populations of self-cleaving molecule.

The activity of the full-length hammerhead ribozyme requires a tertiary interaction between its distal loops leading to the closure of the molecule and its stabilization in the active conformation. In this study, the conformational changes accompanying the cis-cleavage reaction of Chrysanthemum chlorotic mottle viroid hammerhead ribozyme were investigated by high-pressure experiments on the complete cleavage reaction. Two activation volumes (ΔV(≠)) were measured, pointing to the presence of two different populations of molecules corresponding to fast-cleaving and slow-cleaving ribozymes in the reaction mixture. The fast population, with a small ΔV(≠) of 2.6 mL·mol(-1), most likely represents molecules in the near-active conformation, whereas the slow population, with a larger ΔV(≠) of 11.6 mL·mol(-1 , represents molecules that need a larger conformational change to induce activity. In addition, pH-dependence experiments suggest that the group whose deprotonation is required for activity intervenes in the formation of the transition state or in the chemistry of the reaction, but not in the conformational change that precedes it.

Adenine, a hairpin ribozyme cofactor--high-pressure and competition studies.

The RNA world hypothesis assumes that life arose from ancestral RNA molecules, which stored genetic information and catalyzed chemical reactions. Although RNA catalysis was believed to be restricted to phosphate chemistry, it is now established that the RNA has much wider catalytic capacities. In this respect, we devised, in a previous study, two hairpin ribozymes (adenine-dependent hairpin ribozyme 1 and adenine-dependent hairpin ribozyme 2) that require adenine as cofactor for their reversible self-cleavage. We have now used high hydrostatic pressure to investigate the role of adenine in the catalytic activity of adenine-dependent hairpin ribozyme 1. High-pressure studies are of interest because they make it possible to determine the volume changes associated with the reactions, which in turn reflect the conformational modifications and changes in hydration involved in the catalytic mechanism. They are also relevant in the context of piezophilic organisms, as well as in relation to the extreme conditions that prevailed at the origin of life. Our results indicate that the catalytic process involves a transition state whose formation is accompanied by a positive activation volume and release of water molecules. In addition, competition experiments with adenine analogs strongly suggest that exogenous adenine replaces the adenine present at the catalytic site of the wild-type hairpin ribozyme.

[Adaptation of organisms to extreme conditions of deep-sea hydrothermal vents]. / Adaptation des organismes aux conditions extrêmes des sources hydrothermales marines profondes.

The deep-sea hydrothermal vents are located along the volcanic ridges and are characterized by extreme conditions such as unique physical properties (temperature, pression), chemical toxicity, and absence of photosynthesis. However, life exists in these particular environments. The primary producers of energy and organic molecules in these biotopes are chimiolithoautotrophic bacteria. Many animals species live in intimate and complex symbiosis with these sulfo-oxidizing and methanogene bacteria. These symbioses imply a strategy of nutrition and a specific metabolic organization involving numerous interactions and metabolic exchanges, between partners. The organisms of these ecosystems have developed different adaptive strategies. In these environments many microorganisms are adapted to high temperatures. Moreover to survive in these environments, living organisms have developed various strategies to protect themselves against toxic molecules such as H2S and heavy metals.

Hydrostatic and osmotic pressure study of the hairpin ribozyme.

The recent discovery of numerous catalytically active RNAs in various living species as well as the in vitro selection of a large series of RNA aptamers able to bind specifically various molecules such as metabolites and co-factors, emphasize the adaptability of RNAs through the plasticity of their secondary structure. Furthermore, all these observations give support to the "RNA world" hypothesis as a step in the primitive development of life on Earth. On this background, we used high pressure to study the mechanism of action of a model hairpin ribozyme which exhibits self-cleavage and ligation. The activation volume (DeltaV( not equal)) of the cleavage reaction (34+/-4 ml/mol) indicates that an important compaction of the RNA molecule occurs during the reaction and must be accompanied by a significant movement of water molecules . Indeed, such a release of 78+/-4 water molecules per RNA molecule could be measured by complementary osmotic shock experiments. These results are consistent with the information provided by the structural studies which indicate that two loops of the RNA molecule should come into contact for the reaction to occur . The high pressure study of a modified form of the ribozyme whose activity is strictly dependent on the presence of adenine as a co-factor should bring some information about the structural significance of this important DeltaV( not equal) of activation.

Efficient synthesis of fluorothiosparfosic acid analogues with potential antitumoral activity.

In this paper, we describe a short synthesis of N-(phosphonoacetyl)-L-aspartate (PALA) analogues. The mono- and difluorinated thioacetamide precursors were prepared in one step from methyl (diethoxyphosphono)di- and monofluoromethyldithioacetates 8 and 11 as starting materials. Antiproliferating properties on a L1210 strain and ATCase inhibition of these new compounds are disclosed. ThioPALA(FF) 5c showed a remarkable cytotoxic activity towards murine leukemia L1210, when used as tetraester.

The catalytic mechanism of hairpin ribozyme studied by hydrostatic pressure.

The discovery of ribozymes strengthened the RNA world hypothesis, which assumes that these precursors of modern life both stored information and acted as catalysts. For the first time among extensive studies on ribozymes, we have investigated the influence of hydrostatic pressure on the hairpin ribozyme catalytic activity. High pressures are of interest when studying life under extreme conditions and may help to understand the behavior of macromolecules at the origins of life. Kinetic studies of the hairpin ribozyme self-cleavage were performed under high hydrostatic pressure. The activation volume of the reaction (34 +/- 5 ml/mol) calculated from these experiments is of the same order of magnitude as those of common protein enzymes, and reflects an important compaction of the RNA molecule during catalysis, associated to a water release. Kinetic studies were also carried out under osmotic pressure and confirmed this interpretation and the involvement of water movements (78 +/- 4 water molecules per RNA molecule). Taken together, these results are consistent with structural studies indicating that loops A and B of the ribozyme come into close contact during the formation of the transition state. While validating baro-biochemistry as an efficient tool for investigating dynamics at work during RNA catalysis, these results provide a complementary view of ribozyme catalytic mechanisms.

Biochemical and enzymological aspects of the symbiosis between the deep-sea tubeworm Riftia pachyptila and its bacterial endosymbiont.

Riftia pachyptila (Vestimentifera) is a giant tubeworm living around the volcanic deep-sea vents of the East Pacific Rise. This animal is devoid of a digestive tract and lives in an intimate symbiosis with a sulfur-oxidizing chemoautotrophic bacterium. This bacterial endosymbiont is localized in the cells of a richly vascularized organ of the worm: the trophosome. These organisms are adapted to their extreme environment and take advantage of the particular composition of the mixed volcanic and sea waters to extract and assimilate inorganic metabolites, especially carbon, nitrogen, oxygen and sulfur. The high molecular mass hemoglobin of the worm is the transporter for both oxygen and sulfide. This last compound is delivered to the bacterium which possesses the sulfur oxidizing respiratory system, which produces the metabolic energy for the two partners. CO2 is also delivered to the bacterium where it enters the Calvin-Benson cycle. Some of the resulting small carbonated organic molecules are thus provided to the worm for its own metabolism. As far as nitrogen assimilation is concerned, NH3 can be used by the two partners but nitrate can be used only by the bacterium. This very intimate symbiosis applies also to the organization of metabolic pathways such as those of pyrimidine nucleotides and arginine. In particular, the worm lacks the first three enzymes of the de novo pyrimidine biosynthetic pathways as well as some enzymes involved in the biosynthesis of polyamines. The bacterium lacks the enzymes of the pyrimidine salvage pathway. This symbiotic organization constitutes a very interesting system to study the molecular and metabolic basis of biological adaptation.

Integrated allosteric regulation in the S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein.

BACKGROUND: The S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein catalyses the first two reactions of the pyrimidine pathway. In this organism, these two reactions are feedback inhibited by the end product UTP. In the present work, the mechanisms of these integrated inhibitions were studied. RESULTS: The results obtained show that the inhibition is competitive in the case of carbamylphosphate synthetase and non-competitive in the case of aspartate transcarbamylase. They also identify the substrate whose binding is altered by this nucleotide and the step of the carbamylphosphate synthetase reaction which is inhibited. Furthermore, the structure of the domains catalyzing these two reactions were modelled in order to localize the mutations which, specifically, alter the aspartate transcarbamylase sensitivity to the feedback inhibitor UTP. Taken together, the results make it possible to propose a model for the integrated regulation of the two activities of the complex. UTP binds to a regulatory site located in the vicinity of the carbamylphosphate synthetase catalytic subsite which catalyzes the third step of this enzyme reaction. Through a local conformational change, this binding decreases, competitively, the affinity of this site for the substrate ATP. At the same time, through a long distance signal transmission process it allosterically decreases the affinity of the aspartate transcarbamylase catalytic site for the substrate aspartate. CONCLUSION: This investigation provides informations about the mechanisms of allosteric inhibition of the two activities of the CPSase-ATCase complex. Although many allosteric monofunctional enzymes were studied, this is the first report on integrated allosteric regulation in a multifunctional protein. The positions of the point mutations which specifically abolish the sensitivity of aspartate transcarbamylase to UTP define an interface between the carbamylphosphate synthetase and aspartate transcarbamylase domains, through which the allosteric signal for the regulation of aspartate transcarbamylase must be propagated.

Design, synthesis and activity of bisubstrate, transition-state analogues and competitive inhibitors of aspartate transcarbamylase.

Aspartate transcarbamylase initiates the de novo biosynthetic pathway for the production of the pyrimidine nucleotides, precursors of nucleic acids. This pathway is particularly active in rapidly growing cells and tissues. Thus, this enzyme has been tested as a potential target for antiproliferative drugs. In the present work, on the basis of its structural and mechanistic properties, a series of substrate analogues, including potential suicide-pseudosubstrates was synthesized and their putative inhibitory effects were tested using E. coli aspartate transcarbamylase as a model. Two of these compounds appear to be very efficient inhibitors of this enzyme.

Arginine metabolism in the deep sea tube worm Riftia pachyptila and its bacterial endosymbiont.

The present study describes the distribution and properties of enzymes involved in arginine metabolism in Riftia pachyptila, a tubeworm living around deep sea hydrothermal vents and known to be engaged in a highly specific symbiotic association with a bacterium. The results obtained show that the arginine biosynthetic enzymes, carbamyl phosphate synthetase, ornithine transcarbamylase, and argininosuccinate synthetase are present in all of the tissues of the worm and in the bacteria. Thus, Riftia and its bacterial endosymbiont can assimilate nitrogen and carbon via this arginine biosynthetic pathway. The kinetic properties of ornithine transcarbamylase strongly suggest that neither Riftia nor the bacteria possess the catabolic form of this enzyme belonging to the arginine deiminase pathway, the absence of this pathway being confirmed by the lack of arginine deiminase activity. Arginine decarboxylase and ornithine decarboxylase are involved in the biosynthesis of polyamines such as putrescine and agmatine. These activities are present in the trophosome, the symbiont-harboring tissue, and are higher in the isolated bacteria than in the trophosome, indicating that these enzymes are of bacterial origin. This finding indicates that Riftia is dependent on its bacterial endosymbiont for the biosynthesis of polyamines that are important for its metabolism and physiology. These results emphasize a particular organization of the arginine metabolism and the exchanges of metabolites between the two partners of this symbiosis.