Easy Production of "Difficult Peptides" Using Cell-Free Protein Synthesis and a New Methionine Analogue as a Latent Peptide Cleavage Site.

Aliphatic γ-chloro-α-amino acids incorporated in place of their canonical analogues through cell-free protein synthesis act as heat-labile linkers, offering a useful strategy for the straightforward production of target peptides as fusion proteins, from which the targets are readily released. Until now, the natural abundance of aliphatic amino acids in peptides has limited the scope of the method, as it leads to undesired cleavage sites in synthesized products, but here the authors report the development of a new cleavable chloro amino acid that incorporates in place of the relatively rare amino acid methionine, thus greatly expanding the scope of producible targets. This new strategy is employed for simplified peptide synthesis with a methionine-free fusion partner, allowing single-site incorporation of the cleavable linker for clean release and easy purification of the target peptide. Its utility is demonstrated through the straightforward preparation of two peptides reported to be challenging targets and not accessible through standard solid-phase chemical methodologies, as well as analogues.

DNA amplification with in situ nucleoside to dNTP synthesis, using a single recombinant cell lysate of E. coli.

Nucleic acid amplification (NAA) is a cornerstone of modern molecular and synthetic biology. Routine application by non-specialists, however, is hampered by difficulties with storing and handling the requisite labile and expensive reagents, such as deoxynucleoside triphosphates (dNTPs) and polymerases, and the complexity of protocols for their use. Here, a recombinant E. coli extract is reported that provides all the enzymes to support high-fidelity DNA amplification, and with labile dNTPs generated in situ from cheap and stable deoxynucleosides. Importantly, this is obtained from a single, engineered cell strain, through minimal processing, as a lysate capable of replacing the cold-stored commercial reagents in a typical PCR. This inexpensive preparation is highly active, as 1 L of bacterial culture is enough to supply ~106 NAA reactions. Lyophilized lysate can be used after a single-step reconstitution, resulting overall in a greatly simplified workflow and a promising synthetic biology tool, in particular for applications such as diagnostics.

Recombinant cell-lysate-catalysed synthesis of uridine-5'-triphosphate from nucleobase and ribose, and without addition of ATP.

Nucleoside triphosphates (NTPs) are important synthetic targets with diverse applications in therapeutics and diagnostics. Enzymatic routes to NTPs from simple building blocks are attractive, however the cost and complexity of assembling the requisite mixtures of multiple enzymes hinders application. Here, we describe the use of an engineered E. coli cell-free lysate as an efficient readily-prepared multi-enzyme biocatalyst for the production of uridine triphosphate (UTP) from free ribose and nucleobase. Endogenous lysate enzymes are able to support the nucleobase ribosylation and nucleotide phosphorylation steps, while uridine phosphorylation and the production of ribose phosphates (ribose 1-phosphate, ribose 5-phosphate and phosphoribosyl pyrophosphate) require recombinant enrichment of endogenous activities. Co-expression vectors encoding all required recombinant enzymes were employed for host cell transformation, such that a cell-free lysate with all necessary activities was obtained from a single bacterial culture. ATP required as phosphorylation cofactor was recycled by endogenous lysate enzymes using cheap, readily-prepared acetyl phosphate. Surprisingly, acetyl phosphate initiated spontaneous generation of ATP in the lysate, most likely from the breakdown of endogenous pools of adenosine-containing starting materials (e.g. adenosine cofactors, ribonucleic acids). The sub-stoichiometric amount of ATP produced and recycled in this way was enough to support all ATP-dependent steps without addition of any exogenous cofactor or auxiliary enzyme. Using this approach, equimolar solutions of orotic acid and ribose are transformed near quantitatively into 1.4 g L-1 UTP within 2.5 h, using a low-cost, readily-generated biocatalytic preparation.

Exploiting Peptidomimetics to Synthesize Compounds That Activate Ryanodine Receptor Calcium Release Channels.

Ryanodine receptor (RyR) Ca2+ -release channels are essential for contraction in skeletal and cardiac muscle and are prime targets for modification of contraction in disorders that affect either the skeletal or heart musculature. We designed and synthesized a number of compounds with structures based on a naturally occurring peptide (A peptides) that modifies the activity of RyRs. In total, 34 compounds belonging to eight different classes were prepared. The compounds were screened for their ability to enhance Ca2+ release from isolated cardiac sarcoplasmic reticulum (SR) vesicles, with 25 displaying enhanced Ca2+ release. Competition studies with the parent peptides indicated that the synthetic compounds act at a competing site. The activity of the most effective of the compounds, BIT 180, was further explored using Ca2+ release from skeletal SR vesicles and contraction in intact skeletal muscle fibers. The compounds did not alter tension in intact fibers, indicating that (as expected) they are not membrane permeable, but importantly, that they are not toxic to the intact cells. Proof in principal that the compounds would be effective in intact muscle fibers if rendered membrane permeable was obtained with a structurally related membrane-permeable scorpion toxin (imperatoxin A), which was found to enhance contraction.

An unexpected vestigial protein complex reveals the evolutionary origins of an s-triazine catabolic enzyme.

Cyanuric acid is a metabolic intermediate of s-triazines, such as atrazine (a common herbicide) and melamine (used in resins and plastics). Cyanuric acid is mineralized to ammonia and carbon dioxide by the soil bacterium Pseudomonas sp. strain ADP via three hydrolytic enzymes (AtzD, AtzE, and AtzF). Here, we report the purification and biochemical and structural characterization of AtzE. Contrary to previous reports, we found that AtzE is not a biuret amidohydrolase, but instead it catalyzes the hydrolytic deamination of 1-carboxybiuret. X-ray crystal structures of apo AtzE and AtzE bound with the suicide inhibitor phenyl phosphorodiamidate revealed that the AtzE enzyme complex consists of two independent molecules in the asymmetric unit. We also show that AtzE forms an α2ß2 heterotetramer with a previously unidentified 68-amino acid-long protein (AtzG) encoded in the cyanuric acid mineralization operon from Pseudomonas sp. strain ADP. Moreover, we observed that AtzG is essential for the production of soluble, active AtzE and that this obligate interaction is a vestige of their shared evolutionary origin. We propose that AtzEG was likely recruited into the cyanuric acid-mineralizing pathway from an ancestral glutamine transamidosome that required protein-protein interactions to enforce the exclusion of solvent from the transamidation reaction.

Effect of Hydrogen Bonding and Partial Deprotonation on the Oxidation of Peptides.

In a recent computational study, we found that hydrogen bonding/partial deprotonation facilitates subsequent electron transfer from amides to HO•. We have now analyzed these and related reactions with a glycine derivative as a model peptide, investigating not only reaction energies but also barriers for the individual steps. We find that partial deprotonation not only assists subsequent electron transfer (a sequential proton-loss electron-transfer (SPLET)-type reaction pathway) but also promotes sequential hydrogen-atom transfer (HAT, in a sequential proton-loss hydrogen-atom-transfer (SPLHAT)-type process), both being potential alternatives to direct HAT as routes for peptide oxidation. Each of these alternative pathways is calculated to have energy requirements that make them accessible and competitive. These oxidative processes may produce α-carbon-centered peptide radicals that, through deprotonation, are readily oxidized to the corresponding imines. We have also examined the possibility of competing reactions of amino acid side chains by comparing reactions of the glycine model with those of an analogous valine derivative. We find that, while the side chains of amino acids are more reactive toward direct HAT, a preceding partial deprotonation instead continues to favor the SPLET- and SPLHAT-type reactions, leading to the production of α-carbon-centered peptide radicals. Taken together, these processes have broad implications that impact many aspects of the science and utility of peptides.

Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH.

Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from Enterococcus faecalis and Pyrococcus furiosus, the previously unreported enzymes from the hyperthermophiles Thermococcus sibiricus and Thermococcus barophilus, the thermophiles Fervidobacterium nodosum and Thermosipho melanesiensis, and the mesophile Clostridium tetani were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4). Thermococcus barophilus and Thermococcus sibiricus carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the Thermococcus barophilus kinase is efficiently converted in situ to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate.IMPORTANCE Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.

ß-Cyclodextrin- and adamantyl-substituted poly(acrylate) self-assembling aqueous networks designed for controlled complexation and release of small molecules.

Three aqueous self-assembling poly(acrylate) networks have been designed to gain insight into the factors controlling the complexation and release of small molecules within them. These networks are formed between 8.8% 6A-(2-aminoethyl)amino-6A-deoxy-6A-ß-cyclodextrin, ß-CDen, randomly substituted poly(acrylate), PAAß-CDen, and one of the 3.3% 1-(2-aminoethyl)amidoadamantyl, ADen, 3.0% 1-(6-aminohexyl)amidoadamantyl, ADhn, or 2.9% 1-(12-aminododecyl)amidoadamantyl, ADddn, randomly substituted poly(acrylate)s, PAAADen, PAAADhn and PAAADddn, respectively, such that the ratio of ß-CDen to adamantyl substituents is ca. 3:1. The variation of the characteristics of the complexation of the dyes methyl red, methyl orange and ethyl orange in these three networks and by ß-cyclodextrin, ß-CD, and PAAß-CDen alone provides insight into the factors affecting dye complexation. The rates of release of the dyes through a dialysis membrane from the three aqueous networks show a high dependence on host-guest complexation between the ß-CDen substituents and the dyes as well as the structure and the viscosity of the network as shown by ITC, 1H NMR and UV-vis spectroscopy, and rheological studies. Such networks potentially form a basis for the design of controlled drug release systems.

Detection of Biosynthetic Precursors, Discovery of Glycosylated Forms, and Homeostasis of Calcitonin in Human Cancer Cells.

The peptide hormone calcitonin is intimately connected with human cancer development and proliferation. Its biosynthesis is reasoned to proceed via glycine-, α-hydroxyglycine-, glycyllysine-, and glycyllysyllysine-extended precursors; however, as a result of the limitations of current analytical methods, until now, there has been no procedure capable of detecting these individual species in cell or tissue samples. Therefore, their presence and dynamics in cancer had not been established. Here, we report the first methodology for the separation, detection, and quantification of calcitonin and each of its precursors in human cancer cells. We also report the discovery and characterization of O-glycosylated calcitonin and its analogous biosynthetic precursors. Through direct and simultaneous analysis of the glycosylated and nonglycosylated species, we interrogate the hormone biosynthesis. This shows that the cellular calcitonin level is maintained to mitigate effects of biosynthetic enzyme inhibitors that substantially change the proportions of calcitonin-related species released into the culture medium.

Impact of Hydrogen Bonding on the Susceptibility of Peptides to Oxidation.

The tendency of peptides to be oxidized is intimately connected with their function and even their ability to exist in an oxidative environment. Here we report high-level theoretical studies that show that hydrogen bonding can alter the susceptibility of peptides to oxidation, with complexation to a hydrogen-bond acceptor facilitating oxidation, and vice versa, impacting the feasibility of a diverse range of biological processes. It can even provide an energetically viable mechanistic alternative to direct hydrogen-atom abstraction. We find that hydrogen bonding to representative reactive groups leads to a broad (≈400 kJ mol-1 ) spectrum of ionization energies in the case of model amide, thiol and phenol systems. While some of the oxidative processes at the extreme ends of the spectrum are energetically prohibitive, subtle environmental and solvent effects could potentially mitigate the situation, leading to a balance between hydrogen bonding and oxidative susceptibility.

A Peptide Amphiphile Organogelator of Polar Organic Solvents.

A peptide amphiphile is reported, that gelates a range of polar organic solvents including acetonitrile/water, N,N-dimethylformamide and acetone, in a process dictated by ß-sheet interactions and facilitated by the presence of an alkyl chain. Similarities with previously reported peptide amphiphile hydrogelators indicate analogous underlying mechanisms of gelation and structure-property relationships, suggesting that peptide amphiphile organogel design may be predictably based on hydrogel precedents.

ATP Recycling with Cell Lysate for Enzyme-Catalyzed Chemical Synthesis, Protein Expression and PCR.

E. coli lysate efficiently catalyzes acetyl phosphate-driven ATP regeneration in several important biotechnological applications. The utility of this ATP recycling strategy in enzyme-catalyzed chemical synthesis is illustrated through the conversion of uridine to UMP by the lysate from recombinant overexpression of uridine kinase with the E. coli. The UMP is further transformed into UTP through sequential phosphorylations by kinases naturally present in the lysate, in high yield. Cytidine and 5-fluorouridine also give the corresponding NMPs and NTPs with this system. Cell-free protein expression with a processed extract of lysate also proceeds readily when, instead of adding the required NTPs, all four are produced in situ from the NMPs, using acetyl phosphate and relying on endogenous kinase activity. Similarly, dNMPs can be used to produce the dNTPs necessary for DNA synthesis in PCR. These cheap alternative protocols showcase the potential of acetyl phosphate and ATP recycling with readily available cell lysate.

α-Hydrogen Abstraction by •OH and •SH Radicals from Amino Acids and Their Peptide Derivatives.

We have used computational quantum chemistry to investigate the thermochemistry of α-hydrogen abstraction from the full set of amino acids normally found in proteins, as well as their peptide forms, by •OH and •SH radicals. These reactions, with their reasonable complexity in the electronic structure (at the α-carbon), are chosen as a consistent set of models for conducting a fairly robust assessment of theoretical procedures. Our benchmarking investigation shows that, in general, the performance for the various classes of theoretical methods improves in the order nonhybrid DFT → hybrid DFT → double-hybrid DFT → composite procedures. More specifically, we find that the DSD-PBE-P86 double-hybrid DFT procedure yields the best agreement with our high-level W1X-2 vibrationless barriers and reaction energies for this particular set of systems. A significant observation is that, when one considers relative instead of absolute values for the vibrationless barriers and reaction energies, even nonhybrid DFT procedures perform fairly well. To exploit this feature in a cost-effective manner, we have examined a number of multilayer schemes for the calculation of reaction energies and barriers for the abstraction reactions. We find that accurate values can be obtained when a "core" of glycine plus the abstracting radical is treated by DSD-PBE-P86, and the substituent effects are evaluated with M06-2X. Inspection of the set of calculated thermochemical data shows that the correlation between the free energy barriers and reaction free energies is strongest when the reactions are either endergonic or nearly thermoneutral.

Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.

A Cyclodextrin-Based Photoresponsive Molecular Gate that Functions Independently of Either Solvent or Potentially Competitive Guests.

The photoinduced interconversion between cinnamido-substituted cyclodextrins constitutes a gating switch through which the substituent moves to open or block access to the cyclodextrin cavity. Most unusually for a cyclodextrin-based device, the operation of this gate is solvent-independent and unaffected by potentially competitive guests. It occurs in MeOH and DMSO, as well as in water. This contrasts with other cyclodextrin inclusion phenomena that are usually driven by hydrophobic effects and limited to aqueous media.

Outcome-changing effect of polarity reversal in hydrogen-atom-abstraction reactions.

We have examined hydrogen-atom-abstraction reactions for combinations of electrophilic/nucleophilic radicals with electrophilic/nucleophilic substrates. We find that reaction between an electrophilic radical and a nucleophilic substrate is relatively favorable, and vice versa, but the reactions between a radical and a substrate that are both electrophilic or both nucleophilic are relatively unfavorable, consistent with the literature reports of Roberts. As a result, the regioselectivity for the abstraction from a polar substrate can be reversed by reversing the polarity of the attacking radical. Our calculations support Roberts' polarity-reversal-catalysis concept and suggest that addition of a catalyst of appropriate electrophilicity/nucleophilicity can lead to an enhancement of the reaction rate of approximately 5 orders of magnitude. By exploiting the control over regioselectivity associated with the polar nature of the radical and the substrate, we demonstrate the possibility of directing the regioselectivity of hydrogen abstraction from amino acid derivatives and simultaneously providing a significant rate acceleration.

Hydrogen-atom abstraction from a model amino acid: dependence on the attacking radical.

We have used computational chemistry to examine the reactivity of a model amino acid toward hydrogen abstraction by HO•, HOO•, and Br•. The trends in the calculated condensed-phase (acetic acid) free energy barriers are in accord with experimental relative reactivities. Our calculations suggest that HO• is likely to be the abstracting species for reactions with hydrogen peroxide. For HO• abstractions, the barriers decrease as the site of reaction becomes more remote from the electron-withdrawing α-substituents, in accord with a diminishing polar deactivating effect. We find that the transition structures for α- and ß-abstractions have additional hydrogen-bonding interactions, which lead to lower gas-phase vibrationless electronic barriers at these positions. Such favorable interactions become less important in a polar solvent such as acetic acid, and this leads to larger calculated barriers when the effect of solvation is taken into account. For Br• abstractions, the α-barrier is the smallest while the ß-barrier is the largest, with the barrier gradually becoming smaller further along the side chain. We attribute the low barrier for the α-abstraction in this case to the partial reflection of the thermodynamic effect of the captodatively stabilized α-radical product in the more product-like transition structure, while the trend of decreasing barriers in the order ß > γ > δ ∼ ε is explained by the diminishing polar deactivating effect. More generally, the favorable influence of thermodynamic effects on the α-abstraction barrier is found to be smaller when the transition structure for hydrogen abstraction is earlier.

X-ray structure of the amidase domain of AtzF, the allophanate hydrolase from the cyanuric acid-mineralizing multienzyme complex.

The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.

Biocatalysis for the application of CO2 as a chemical feedstock.

Biocatalysts, capable of efficiently transforming CO2 into other more reduced forms of carbon, offer sustainable alternatives to current oxidative technologies that rely on diminishing natural fossil-fuel deposits. Enzymes that catalyse CO2 fixation steps in carbon assimilation pathways are promising catalysts for the sustainable transformation of this safe and renewable feedstock into central metabolites. These may be further converted into a wide range of fuels and commodity chemicals, through the multitude of known enzymatic reactions. The required reducing equivalents for the net carbon reductions may be drawn from solar energy, electricity or chemical oxidation, and delivered in vitro or through cellular mechanisms, while enzyme catalysis lowers the activation barriers of the CO2 transformations to make them more energy efficient. The development of technologies that treat CO2-transforming enzymes and other cellular components as modules that may be assembled into synthetic reaction circuits will facilitate the use of CO2 as a renewable chemical feedstock, greatly enabling a sustainable carbon bio-economy.

Hydrogen from formic acid through its selective disproportionation over sodium germanate--a non-transition-metal catalysis system.

A robust catalyst for the selective dehydrogenation of formic acid to liberate hydrogen gas has been designed computationally, and also successfully demonstrated experimentally. This is the first such catalyst not based on transition metals, and it exhibits very encouraging performance. It represents an important step towards the use of renewable formic acid as a hydrogen-storage and transport vector in fuel and energy applications.