Identification of the yeast 20S proteasome catalytic centers and subunit interactions required for active-site formation

The proteasome is responsible for degradation of substrates of the ubiquitin pathway. 20S proteasomes are cylindrical particles with subunits arranged in a stack of four heptameric rings. The outer rings are composed of α subunits, and the inner rings are composed of β subunits. A well-characterized archaeal proteasome has a single type of each subunit, and the N-terminal threonine of the β subunit is the active-site nucleophile. Yeast proteasomes have seven different β subunits and exhibit several distinct peptidase activities, which were proposed to derive from disparate active sites. We show that mutating the N-terminal threonine in the yeast Pup1 β subunit eliminates cleavage after basic residues in peptide substrates, and mutating the corresponding threonine of Pre3 prevents cleavage after acidic residues. Surprisingly, neither mutation has a strong effect on cell growth, and they have at most minor effects on ubiquitin-dependent proteolysis. We show that Pup1 interacts with Pup3 in each β subunit ring. Our data reveal that different proteasome active sites contribute very differently to protein breakdown in vivo, that contacts between particular subunits in each β subunit ring are critical for active-site formation, and that active sites in archaea and different eukaryotes are highly similar.

UV light selectively coinduces supply pathways from primary metabolism and flavonoid secondary product formation in parsley

The UV light-induced synthesis of UV-protective flavonoids diverts substantial amounts of substrates from primary metabolism into secondary product formation and thus causes major perturbations of the cellular homeostasis. Results from this study show that the mRNAs encoding representative enzymes from various supply pathways are coinduced in UV-irradiated parsley cells (Petroselinum crispum) with two mRNAs of flavonoid glycoside biosynthesis, encoding phenylalanine ammonia-lyase and chalcone synthase. Strong induction was observed for mRNAs encoding glucose 6-phosphate dehydrogenase (carbohydrate metabolism, providing substrates for the shikimate pathway), 3-deoxyarabinoheptulosonate 7-phosphate synthase (shikimate pathway, yielding phenylalanine), and acyl-CoA oxidase (fatty acid degradation, yielding acetyl-CoA), and moderate induction for an mRNA encoding S-adenosyl-homocysteine hydrolase (activated methyl cycle, yielding S-adenosyl-methionine for B-ring methylation). Ten arbitrarily selected mRNAs representing various unrelated metabolic activities remained unaffected. Comparative analysis of acyl-CoA oxidase and chalcone synthase with respect to mRNA expression modes and gene promoter structure and function revealed close similarities. These results indicate a fine-tuned regulatory network integrating those functionally related pathways of primary and secondary metabolism that are specifically required for protective adaptation to UV irradiation. Although the response of parsley cells to UV light is considerably broader than previously assumed, it contrasts greatly with the extensive metabolic reprogramming observed previously in elicitor-treated or fungus-infected cells.

Unusual 1H NMR chemical shifts support (His) Cɛ1—H⋅⋅⋅O⩵C H-bond: Proposal for reaction-driven ring flip mechanism in serine protease catalysis

13C-selective NMR, combined with inhibitor perturbation experiments, shows that the Cɛ1—H proton of the catalytic histidine in resting α-lytic protease and subtilisin BPN′ resonates, when protonated, at 9.22 ppm and 9.18 ppm, respectively, which is outside the normal range for such protons and ≈0.6 to 0.8 ppm further downfield than previously reported. They also show that the previous α-lytic protease assignments [Markley, J. L., Neves, D. E., Westler, W. M., Ibanez, I. B., Porubcan, M. A. & Baillargeon, M. W. (1980) Front. Protein Chem. 10, 31–61] were to signals from inactive or denatured protein. Simulations of linewidth vs. pH demonstrate that the true signal is more difficult to detect than corresponding signals from inactive derivatives, owing to higher imidazole pKa values and larger chemical shift differences between protonated and neutral forms. A compilation and analysis of available NMR data indicates that the true Cɛ1—H signals from other serine proteases are similarly displaced downfield, with past assignments to more upfield signals probably in error. The downfield displacement of these proton resonances is shown to be consistent with an H-bond involving the histidine Cɛ1—H as donor, confirming the original hypothesis of Derewenda et al. [Derewenda, Z. S., Derewenda, U. & Kobos, P. M. (1994) J. Mol. Biol. 241, 83–93], which was based on an analysis of literature x-ray crystal structures of serine hydrolases. The invariability of this H-bond among enzymes containing Asp-His-Ser triads indicates functional importance. Here, we propose that it enables a reaction-driven imidazole ring flip mechanism, overcoming a major dilemma inherent in all previous mechanisms, namely how these enzymes catalyze both the formation and productive breakdown of tetrahedral intermediates.

Contribution of Salmonella typhimurium type III secretion components to needle complex formation

The prgHIJK operon encodes components of the Salmonella typhimurium pathogenicity island 1 type III secretion system (TTSS). Previously, prgH and prgK were shown to be required for formation of the supramolecular type III secretion needle complex (NC) [Kubori, T., et al. (1998) Science 280, 602–605]. This work indicates that all prg operon genes are required for NC formation. PrgH multimerizes into a distinct tetrameric-shaped structure that may be an early intermediate of NC assembly and may provide the structural foundation required for PrgK oligomerization. PrgH and PrgK, in the absence of other TTSS components, oligomerize into ring-shaped structures identical in appearance and size to the base of the NC, indicating that they are likely the major inner membrane structural components required for secretion. PrgI and PrgJ cofractionate with the NC and are secreted into the culture supernatant. NC from prgI and prgJ mutants have an identical morphology to the envelope-spanning (basal body) NC components, but are missing the external needle, indicating that PrgI and PrgJ are required for full NC assembly and are likely components of the external needle. Therefore, PrgI and PrgJ are secreted through the NC basal body, composed in part of PrgH/K and InvG/H rings, to participate in assembly of the more distal components of the NC.

Incipient species formation in salamanders of the Ensatina complex

The Ensatina eschscholtzii complex of plethodontid salamanders, a well-known “ring species,” is thought to illustrate stages in the speciation process. Early research, based on morphology and coloration, has been extended by the incorporation of studies of protein variation and mitochondrial DNA sequences. The new data show that the complex includes a number of geographically and genetically distinct components that are at or near the species level. The complex is old and apparently has undergone instances of range contraction, isolation, differentiation, and then expansion and secondary contact. While the hypothesis that speciation is retarded by gene flow around the ring is not supported by molecular data, the general biogeographical hypothesis is supported. There is evidence of a north to south range expansion along two axes, with secondary contact and completion of the ring in southern California. Current research targets regions once thought to show primary intergradation, but which molecular markers reveal to be zones of secondary contact. Here emphasis is on the subspecies E. e. xanthoptica, which is involved in four distinct secondary contacts in central California. There is evidence of renewed genetic interactions upon recontact, with greater genetic differentiation within xanthoptica than between it and some of the interacting populations. The complex presents a full array of intermediate conditions between well-marked species and geographically variable populations. Geographically differentiated segments represent a diversity of depths of time of isolation and admixture, reflecting the complicated geomorphological history of California. Ensatina illustrates the continuing difficulty in making taxonomic assignments in complexes studied during species formation.

Biochemical evolution III: Polymerization on organophilic silica-rich surfaces, crystal–chemical modeling, formation of first cells, and geological clues

Catalysis at organophilic silica-rich surfaces of zeolites and feldspars might generate replicating biopolymers from simple chemicals supplied by meteorites, volcanic gases, and other geological sources. Crystal–chemical modeling yielded packings for amino acids neatly encapsulated in 10-ring channels of the molecular sieve silicalite-ZSM-5-(mutinaite). Calculation of binding and activation energies for catalytic assembly into polymers is progressing for a chemical composition with one catalytic Al–OH site per 25 neutral Si tetrahedral sites. Internal channel intersections and external terminations provide special stereochemical features suitable for complex organic species. Polymer migration along nano/micrometer channels of ancient weathered feldspars, plus exploitation of phosphorus and various transition metals in entrapped apatite and other microminerals, might have generated complexes of replicating catalytic biomolecules, leading to primitive cellular organisms. The first cell wall might have been an internal mineral surface, from which the cell developed a protective biological cap emerging into a nutrient-rich “soup.” Ultimately, the biological cap might have expanded into a complete cell wall, allowing mobility and colonization of energy-rich challenging environments. Electron microscopy of honeycomb channels inside weathered feldspars of the Shap granite (northwest England) has revealed modern bacteria, perhaps indicative of Archean ones. All known early rocks were metamorphosed too highly during geologic time to permit simple survival of large-pore zeolites, honeycombed feldspar, and encapsulated species. Possible microscopic clues to the proposed mineral adsorbents/catalysts are discussed for planning of systematic study of black cherts from weakly metamorphosed Archaean sediments.

Transition metal ion activation of DNA binding by the diphtheria tox repressor requires the formation of stable homodimers.

The diphtheria tox repressor (DtxR) is a transition metal ion-dependent regulatory element that controls the expression of diphtheria toxin and several genes involved in the synthesis of siderophores in Corynebacterium diphtheriae. In the presence of transition metal ions apo-DtxR becomes activated and specifically binds to its target DNA sequences. We demonstrate by glutaraldehyde cross-linking that monomeric apo-DtxR is in weak equilibrium with a dimeric form and that upon addition of activating metal ions to the reaction mixture a dimeric complex is stabilized. Addition of the DNA-binding-defective mutant apo-DtxR(delta 1-47) to apo-DtxR in the absence of transition metal ions inhibits conversion of the apo-repressor to its activated DNA-binding form. We also show that the binding of Ni2+ to both apo-DtxR and apo-DtxR(delta 1-47) is cooperative and that upon ion binding there is a conformational change in the environment of the indole ring moiety of Trp-104. For the wild-type repressor the consequences of this conformational change include a shift in equilibrium toward dimer formation and activation of target DNA binding by the repressor. We conclude that the formation of DtxR homodimers is mediated through a protein-protein interaction domain that is also activated on metal ion binding.

Missing Rings in Pinus halepensis – The Missing Link to Relate the Tree-Ring Record to Extreme Climatic Events

Climate predictions for the Mediterranean Basin include increased temperatures, decreased precipitation, and increased frequency of extreme climatic events (ECE). These conditions are associated with decreased tree growth and increased vulnerability to pests and diseases. The anatomy of tree rings responds to these environmental conditions. Quantitatively, the width of a tree ring is largely determined by the rate and duration of cell division by the vascular cambium. In the Mediterranean climate, this division may occur throughout almost the entire year. Alternatively, cell division may cease during relatively cool and dry winters, only to resume in the same calendar year with milder temperatures and increased availability of water. Under particularly adverse conditions, no xylem may be produced in parts of the stem, resulting in a missing ring (MR). A dendrochronological network of Pinus halepensis was used to determine the relationship of MR to ECE. The network consisted of 113 sites, 1,509 trees, 2,593 cores, and 225,428 tree rings throughout the distribution range of the species. A total of 4,150 MR were identified. Binomial logistic regression analysis determined that MR frequency increased with increased cambial age. Spatial analysis indicated that the geographic areas of south-eastern Spain and northern Algeria contained the greatest frequency of MR. Dendroclimatic regression analysis indicated a non-linear relationship of MR to total monthly precipitation and mean temperature. MR are strongly associated with the combination of monthly mean temperature from previous October till current February and total precipitation from previous September till current May. They are likely to occur with total precipitation lower than 50 mm and temperatures higher than 5°C. This conclusion is global and can be applied to every site across the distribution area. Rather than simply being a complication for dendrochronology, MR formation is a fundamental response of trees to adverse environmental conditions. The demonstrated relationship of MR formation to ECE across this dendrochronological network in the Mediterranean basin shows the potential of MR analysis to reconstruct the history of past climatic extremes and to predict future forest dynamics in a changing climate.

Different behavior of nitrenes and carbenes on photolysis and thermolysis: Formation of azirine, ylidic cumulene, and cyclic ketenimine and the rearrangement of 6-phenanthridylcarbene to 9-phenanthrylnitrene

Flash vacuum thermolysis (FVT) of 9-azidophenanthrene 8, 6-(5-tetrazolyl)phenanthridine 18, and [1,2,3]triazolo[1,5-f]phenanthridine 19 yields 9-cyanofluorene 12 as the principal product and 4-cyanofluorene as a minor product. In all cases, when the product is condensed at or below 77 K, the seven-membered ring ketenimine 24 is detectable by IR spectroscopy (1932 cm(-1)) up to 200 K. Photolysis of Ar matrix isolated 8 at lambda = 308 or 313 nm generates at first the azirine 26, rapidly followed by the ylidic cumulene 27. The latter reverts to azirine 26 at lambda > 405 nm, and the azirine reverts to the ylidic cumulene at 313 nm. Nitrene 9 is observed by ESR spectroscopy following FVT of either azide 8, tetrazole 18, or triazole 19 with Ar matrix isolation of the products. Nitrene 9 and carbene 21 are observed by ESR spectroscopy in the Ar matrix photolyses of azide 8 and triazole 19, respectively.

NGC922 - a New Drop-through Ring Galaxy

We have found the peculiar galaxy NGC 922 to be a new drop-through ring galaxy using multiwavelength (ultraviolet-radio) imaging and spectroscopic observations. Its 'C'-shaped morphology and tidal plume indicate a recent strong interaction with its companion which was identified with these observations. Using numerical simulations we demonstrate that the main properties of the system can be generated by a high-speed off-axis drop-through collision of a small galaxy with a larger disc system, thus making NGC 922 one of the nearest known collisional ring galaxies. While these systems are rare in the local Universe, recent deep Hubble Space Telescope images suggest they were more common in the early Universe.

The cationic ring-opening polymerization of cyclic ethers

The kinetics and mechanisms of ring opening polymerization and copolymerizntion of different cyclic ethers were studied using mainly a cationic system of iinitiation. BF30Et2/ethanediol. The cyclic ethers reacted differently showing that ring strain and basicity are the main driving forces in cationic ring opening polymerizaion. In most cases it was found that the degree of polymerization is controlled kinetically via terminations with the counterion and the monomers, and that the contribution of each type of reaction to the overall termination differs markedly. The Gel permeation chromatography studies showed that the molecular weight distribution of the samples of polyoxetanes were bimodal. This was in accordance with previous work establishing that the cyclic tetramer is found in much higher proportions than any of the other cyclic oligomers. However the molecular weight distribution of the copolymers made from oxetane and THF or from oxetane and oxepane were shown to be unimodal. These observations could be explained by a change in the structure of the growing end involved in the cationic polymerization. In addition crown ethers like dibenzo-crown-6 and compounds such as veratrole are believed to stabilise the propagating end and promote the formation of living polymers from oxetane.

The ring opening polymerization of ring strained cyclic ethers

The kinetics and mechanisms of the ring-opening polymerization of oxetane were studied using cationic and coordinated anionic catalysts. The cationic initiators used were BF30Et2!/ethanol, BF30Et2!/ethanediol and BF30Et2/propantriol. Kinetic determinations with the BF30Et2/diol system indicated that a 1: 1 BF3:0H ratio gave the maximum rate of polymerization and this ratio was employed to detenmne the overall rates of polymerization. An overall second-order dependence was obtained when the system involved ethanediol or propantriol as co-catalyst and a 3/2-order dependence with ethanol, in each case the monomer gave a first-order relationship. This suggested that two mechanisms accounted for the cationic polymerization. These mechanisms were investigated and further evidence for these was obtained from the study of the complex formation of BF30Et2 and the co-catalysts by 1H NMR. Molecular weight studies (using size-exclusion chromatography) indicated that the hydroxyl ion acted as a chain transfer reagent when the [OH] > [BF3]. A linear relationship was observed when the number average molecular weight was plotted against [oxetane] at constant [BF3:0H], and similarly a linear dependency was observed on the BF3:0H 1:1 adduct at constant oxetane concentration. Copolymerization of oxetane and THF was carried out using BF30Et2/ethanol system. The reactivity ratios were calculated as rOXT = 1.2 ± 0.30 and rTHF = 0.14 ± 0.03. These copolymers were random copolymers with no evidence of oligomer formation. The coordinated anionic catalyst, porphinato-aluminium chloride [(TPP)AICl], was used to produce a living polymerization of oxetane. An overall third-order kinetics was obtained, with a second-order with respect to the [(TPP)AICl] and a first-order with respect to the [oxetane] and a mechanism was postulated using these results. The stereochemistry of [(TPP)AlCl] catalyst was investigated using cyclohexene and cyclopentene oxide monomers, using extensive 1H NMR, 2-D COSY and decoupling NMR techniques it was concluded that [(TPP)AlCl] gave rise to stereoregular polymers.

Cationic ring opening polymerisation (CROP) of oxetane and its derivatives using oxonium derived initiators

Several cationic initiator systems were developed and used to polymerise oxetane with two oxonium ion initiator systems being investigated in depth. The first initiator system was generated by the elimination of a chloride group from a chloro methyl ethyl ether. Adding a carbonyl co-catalyst to a carbocationic centre generated the second initiator system. It was found that the anion used to stabilise the initiator was critical to the initial rate of polymerisation of oxetane with hexafluoroantimonate resulting in the fastest polymerisations. Both initiator systems could be used at varying monomer to initiator concentrations to control the molecular number average, Mn, of the resultant polymer. Both initiator systems showed living characteristics and were used to polymerise further monomers and generate higher molecular weight material and block copolymers. Oxetane and 3,3-dimethyl oxetane can both be polymerised using either oxonium ion initiator system in a variety of DCM or DCM/1,4-dioxane solvent mixtures. The level of 1,4-dioxane does have an impact on the initial rate of polymerisation with higher levels resulting in lower initial rates of polymerisation but do tend to result in higher polydispersities. The level of oligomer formation is also reduced as the level of 1,4-dioxane is increased. 3,3-bis-bromomethyl oxetane was also polymerised but a large amount of hyperbranching was seen at the bromide site resulting in a difficult to solvate polymer system. Multifunctional initiator systems were also generated using the halide elimination reactions with some success being achieved with 1,3,5-tris-bromomethyl-2,4,6-tris-methyl-benzene derived initiator system. This offered some control over the molecular number average of the resultant polymer system.

Catalyst studies on the ring opening of tetrahydrofuran-dimethanol to 1,2,6-hexanetriol

The metal catalyzed hydrogenolysis of the biomass-derived THF-dimethanol to 1,2,6-hexanetriol using heterogeneous catalysts was investigated. Bimetallic Rh-Re catalysts (4 wt% Rh and a Re/Rh (mol. ratio of 0.5) on a silica support gave the best performance and 1,2,6-hexanetriol was obtained in 84% selectivity at 31% conversion (120 C, 80 bar, 4 h); the selectivity reaches a maximum of 92% at 80 C. The product distribution at prolonged reaction times or higher temperatures or both shows the formation of diols and mono-alcohols, indicating that the 1,2,6-hexanetriol is prone to subsequent hydrodeoxygenation reactions. Different silica supports were investigated and optimal results were obtained with an amorphous silica featuring an intermediate surface area and an average mesopore size of about 6 nm. TPR and XPS surface analysis support the presence of mixed Rh and Re particles. The redox Reδ+/ReTotal surface ratio correlates with the conversion in a volcano type dependency. Both gas phase as well as Rh200Re1OH cluster DFT calculations support an acid-metal bifunctional mechanism and explain the products distribution. © 2013 Elsevier B.V. All rights reserved.

Formation of Polycyclic Aromatic Hydrocarbons and Nitrogen Containing Polycyclic Aromatic Compounds in Titan's Atmosphere, the Interstellar Medium and Combustion

Several different mechanisms leading to the formation of (substituted) naphthalene and azanaphthalenes were examined using theoretical quantum chemical calculations. As a result, a series of novel synthetic routes to Polycyclic Aromatic Hydrocarbons (PAHs) and Nitrogen Containing Polycyclic Aromatic Compounds (N-PACs) have been proposed. On Earth, these aromatic compounds originate from incomplete combustion and are released into our environment, where they are known to be major pollutants, often with carcinogenic properties. In the atmosphere of a Saturn's moon Titan, these PAH and N-PACs are believed to play a critical role in organic haze formation, as well as acting as chemical precursors to biologically relevant molecules. The theoretical calculations were performed by employing the ab initio G3(MP2,CC)/B3LYP/6-311G** method to effectively probe the Potential Energy Surfaces (PES) relevant to the PAH and N-PAC formation. Following the construction of the PES, Rice-Ramsperger-Kassel-Markus (RRKM) theory was used to evaluate all unimolecular rate constants as a function of collision energy under single-collision conditions. Branching ratios were then evaluated by solving phenomenological rate expressions for the various product concentrations. The most viable pathways to PAH and N-PAC formation were found to be those where the initial attack by the ethynyl (C2H) or cyano (CN) radical toward a unsaturated hydrocarbon molecule led to the formation of an intermediate which could not effectively lose a hydrogen atom. It is not until ring cyclization has occurred, that hydrogen elimination leads to a closed shell product. By quenching the possibility of the initial hydrogen atom elimination, one of the most competitive processes preventing the PAH or N-PAC formation was avoided, and the PAH or N-PAC formation was allowed to proceed. It is concluded that these considerations should be taken into account when attempting to explore any other potential routes towards aromatic compounds in cold environments, such as on Titan or in the interstellar medium.