Capping Reactions Research Articles

Intramolecular carbon isotope geochemistry of butane isomers from laboratory maturation and Monte-Carlo simulations of kerogen types I, II, and III

Position-specific (PS) carbon isotope compositions of light hydrocarbons such as propane and butane isomers (n-butane and i-butane) can provide a wealth of information on the history of natural gases in the subsurface reservoirs and other environments. For PS carbon isotope analysis of butane isomers, we have established a GC-pyrolysis-GC-isotope ratio mass spectrometry method with demonstrated accuracy. With this method, we analyzed PS δ13C values of butane isomers generated from the systematic laboratory pyrolysis experiments of three different kerogen types (I, II, and III) at temperatures of 310–430 °C with corresponding thermal maturity (Easy %Ro) ranging from 0.7 to 3.3. The observed evolution in the abundances of butane isomers can be interpreted and semi-quantitatively modeled based on the abundances of different CC bonds within the kerogens at low maturity and thermal degradation of butane isomers at high maturity. The δ13C values at the central sites of both nC4 and iC4 were heavier than those at the terminal positions, similar to our previous observations of propane. Their isotopic evolution with the maturity were controlled largely by kinetic isotope effects associated with breaking of different CC bonds during the generation and degradation of butane isomers. Kinetic Monte Carlo (kMC) simulations of n-butane generated from thermal cracking of model kerogens (I, II, and III) and an oil with a series of reactions (homolytic cleavage, β-scission, radical isomerization, H-abstraction, and termination by radical recombination) provided generally consistent results with the experimental observations, although the difference in PS δ13C values between the central and terminal positions are somewhat overestimated. On the other hand, the kMC simulation with homolytic cleavage and capping reactions alone produced significant deviations from the experimental results. Re-assessment of very limited data of PS δ13C values of natural butanes with our experimental and simulation results show that biodegradation significantly increased δ13C values at the central positions, not only of propane, but also of both butane isomers. This study lays a foundation and demonstrates the potential of PS isotope geochemistry of butane isomers to further improve our understanding of the sources, and geochemical and microbial processes of light hydrocarbons in the subsurface and other natural environments.

Investigation of the effect of 5’UTR on immunogenicity in coxsackievirus VP1 mRNA vaccine

Abstract Hand, foot and mouth disease (HFMD) is a group of common infectious diseases caused by various enteroviruses, which are transmitted through gastrointestinal tract, respiratory tract and contact. The mRNA vaccine is safe, effective, simple to produce and easy to promote, and has attracted much attention in recent years. Starting from the screening modification of the 5’UTR of antigen nucleic acid sequence, the mRNA translation efficiency was improved by introducing the antigen epitope cleavage processing signal through the untranslated region screening modification, and the nucleic acid vaccine design with stable translation characteristics and effective control of inflammation homeostasis was studied to provide a reference for the molecular design of future mRNA vaccines. In this study, 5’UTRs of different lengths of human β-globin origin were successfully constructed in front of the gene sequences of CA6-VP1 and CA10-VP1; the target gene sequences were transcribed in vitro with capping and tailing reactions to obtain structurally complete mRNA sequences; 293T cells were transfected and Western Blot assays were performed to find that the target genes with the modified 5’UTRs attached The mRNA was able to express the protein correctly and stably, and the protein expression was more compared with the original sequence without the modified 5’UTR. The human β-globin 5’UTR is useful for improving the translation efficiency and protein stability of the target gene mRNA. Further experimental evidence is needed to assess its induction of immune responses in vivo.

On the Mechanism of Soot Nucleation. IV. Molecular Growth of the Flattened E-Bridge.

Rotationally excited dimerization of aromatic moieties, a mechanism proposed recently to explain the initial steps of soot particle inception in combustion and pyrolysis of hydrocarbons, produces a molecular structure, termed E-bridge, combining the two aromatics via five-membered aromatic rings sharing a common bond. The present study investigates a hydrogen-mediated addition of acetylene to the fused five-membered ring part of the E-bridge forming a seven-membered ring. The carried out quantum-mechanical and rate theoretical calculations indicate the plausibility of such capping reactions, and kinetic Monte Carlo simulations demonstrate their frequent occurrence. The capping frequency, however, is limited by "splitting" the fused five-membered bridge due to five-membered ring migration. A similar migration of edge seven-membered rings is shown to be also rapid but short, as their encounter with five-membered rings converts them both into six-membered rings.

Bay capping via acetylene addition to polycyclic aromatic hydrocarbons: Mechanism and kinetics

The bay-capping mechanism on PAH armchair edges and the kinetics of acetylene addition to 6–6–5 and 5–6–5 bays have been explored by ab initio/RRKM-ME calculations. The bays on the edges were modeled by C21H11 and C20H9 radicals produced by H abstractions from 7H-benzo[c]cyclopenta[e]pyrene and dicyclopenta[cf]pyrene. The C20H9 + C2H2 reaction is shown to have a low entrance barrier and to rapidly form the capped product, indaceno[2,1,8,7-cdefg]pyrene, along with ethynyl substituted dicyclopenta[cf]pyrene at temperatures above 1400 K. The reactivity of C21H11 is shown to be governed by the location of the unpaired electron; the π radical R1 formed by H abstraction from the CH2 group in 7H-benzo[c]cyclopenta[e]pyrene reacts with C2H2 very slowly owing to a high entrance barrier, with the bay-capping rate constant approaching 10−16 cm3 molecule−1 s−1 only at temperatures above 2000 K. This result reaffirms that the growth of π aryl radicals via acetylene addition is inefficient and reflects the generally low reactivity of such radicals where the spin density is highly delocalized over the entire polyaromatic system. Alternatively, the σ C21H11 radical R2 produced by H abstraction from the five-membered ring at the bay rapidly reacts with C2H2 forming the bay-capped product, with the rate constant on the order of 10−12 cm3 molecule−1 s−1 at T ≥ 1500 K. Rate constants for the capping reactions at the 6–6–5 and 5–6–5 bays are compared with those at the 6–0–6, 6–6–6, and 6–5–6 bays. The site-specific bay-capping rate constants have been utilized in kMC simulations of the PAH growth and the results showed measurable differences when the 6–6–5 and 5–6–5 bay-capping reactions are taken into account, including an increase of the growth rate and the formation of closed-shell PAH and a rise of the number of embedded five-membered rings accompanied with a slight decrease of their overall amount.

Characterisation of ZnO nanoparticles prepared using aqueous leaf extracts of Chromolaena odorata (L.) and Manihot esculenta (Crantz)

Plant-mediated routes for synthesising metal oxide nanoparticles are gaining tremendous attention due to the benefits of the technique: simplicity, cost-effectiveness, and eco-friendliness. We compared the properties of zinc oxide nanoparticles (ZnONPs) made from aqueous leaf extracts of Chromolaena odorata and Manihot esculenta, both of which are abundant on the African continent. The phytochemical composition of the extracts was first assessed using gas chromatography-mass spectrometry (GC-MS) to determine the types of biomolecules involved in the reducing and capping processes that result in ZnONP formation. After that, UV-Vis spectrophotometry, scanning electron microscopy, energy dispersive X-ray analysis, transmission electron microscopy, X-ray diffractometry, and Fourier transform infrared spectroscopy (FTIR) were used to study ZnONP formation, morphological characteristics, elemental composition, shape and size properties, and phase composition. The ZnONPs made with Chromolaena odorata leaf extract had a better distribution of spherical and hexagonal forms, with an average particle size of 42.35 nm. The ZnONPs made with Manihot esculenta leaf as a reductant had a particle size of 14.71 nm on average and were more agglomerated with poor particle distribution. Phytosterols were shown to be the most important biomolecules in the reduction and capping reactions, according to GC-MS and FTIR analyses. In this study, we created a cost-effective technique for the synthesis of eco-friendly ZnONPs for diverse applications, particularly in Africa, using Chromolaena odorata and Manihot esculenta leaves.Significance: This study could provide useful information on how the phytochemicals embedded in Chromolaena odorata and Manihot esculenta could influence the properties of the ZnONPs obtained from them. Differences in morphology and formation yield of ZnONPs are obtainable from aqueous leaf extracts of Chromolaena odorata and Manihot esculenta. Chromolaena odorata and Manihot esculenta could serve as dependable raw materials for the green synthesis of ZnONPs in Africa.

Rhodocenium Functionalization Enabled by Half-Sandwich Capping, Zincke Reaction, Diazoniation and Sandmeyer Chemistry.

In continuation of our exploration of metallocenium chemistry we report here on innovative ways toward monofunctionalized rhodocenium salts applying half‐sandwich capping reactions of cyclopentadienyl rhodium(III) halide synthons with cyclopentadienyl ylides containing pyridine, phosphine or dinitrogen leaving groups, followed by Zincke and Sandmeyer reactions. Thereby amino, diazonio, bromo, azido and iodo rhodocenium salts containing valuable functional groups are accessible for the first time. Target compounds were characterized by spectroscopic (1H/13C/103Rh‐NMR, IR, HR‐MS), structural (single crystal XRD) and electrochemical (CV) methods and their properties were compared to those of isoelectronic cobaltocenium compounds. These new functionalized rhodocenium complexes significantly expand the so far extremely limited chemical space of rhodocenium salts with promising options for the future development in the area of rhodocenium chemistry.

High resolution biosensor to test the capping level and integrity of mRNAs.

5′ Cap structures are ubiquitous on eukaryotic mRNAs, essential for post-transcriptional processing, translation initiation and stability. Here we describe a biosensor designed to detect the presence of cap structures on mRNAs that is also sensitive to mRNA degradation, so uncapped or degraded mRNAs can be detected in a single step. The biosensor is based on a chimeric protein that combines the recognition and transduction roles in a single molecule. The main feature of this sensor is its simplicity, enabling semi-quantitative analyses of capping levels with minimal instrumentation. The biosensor was demonstrated to detect the capping level on several in vitro transcribed mRNAs. Its sensitivity and dynamic range remained constant with RNAs ranging in size from 250 nt to approximately 2700 nt and the biosensor was able to detect variations in the capping level in increments of at least 20%, with a limit of detection of 2.4 pmol. Remarkably, it also can be applied to more complex analytes, such mRNA vaccines and mRNAs transcribed in vivo. This biosensor is an innovative example of a technology able to detect analytically challenging structures such as mRNA caps. It could find application in a variety of scenarios, from quality analysis of mRNA-based products such as vaccines to optimization of in vitro capping reactions.

Kinetic Monte Carlo statistics of curvature integration by HACA growth and bay closure reactions for PAH growth in a counterflow diffusion flame

This paper uses a Kinetic Monte Carlo model that includes processes to integrate curvature due to the formation of five- and seven-member rings to simulate polycyclic aromatic hydrocarbons (PAHs) growing in lightly sooting ethylene and acetylene counterflow diffusion flames. The model includes new processes to form seven-member rings via hydrogen-abstraction-acetylene-addition and bay closure reactions on sites containing partially embedded five-member rings. The model additionally includes bay closure and HACA bay capping reactions for the integration of five-member rings. The mass spectra of PAHs predicted by the model are assessed against experimental data, and the distribution of embedded five-member rings and seven-member rings is studied as a function of spatial location, molecule size and frequency of events sampled in the simulation. The simulations show that the formation of seven-member rings and the embedding of five-member rings is a competitive process. Both types of rings are observed more frequently as the simulation proceeds from the fuel outlet towards the stagnation plane. Approximately 15% of the events that integrate curvature resulted in the formation of a seven-member ring coupled to an embedded five-member ring, and the remaining 85% of events embedded five-member rings via the formation of six-member rings. The proportion of PAHs containing embedded five-member rings and/or seven-member rings is observed to be a function of PAH size, passing through a maximum for PAHs containing 15–20 six-member rings. However, the proportion of PAHs containing both types of ring increases with PAH size, where upwards of 10% of PAHs containing at least one five-member ring and 15 or more six-member rings also contain a seven-member ring.

Characterization of 3'-Phosphate RNA Ligase Paralogs RtcB1, RtcB2, and RtcB3 from Myxococcus xanthus Highlights DNA and RNA 5'-Phosphate Capping Activity of RtcB3.

Escherichia coli RtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3'-PO4 ends to 5'-OH ends via stable RtcB-(histidinyl-N)-GMP and transient RNA3'pp5'G intermediates. E. coli RtcB also transfers GMP to a DNA 3'-PO4 end to form a stable "capped" product, DNA3'pp5'G. RtcB homologs are found in a multitude of bacterial proteomes, and many bacteria have genes encoding two or more RtcB paralogs; an extreme example is Myxococcus xanthus, which has six RtcBs. In this study, we purified, characterized, and compared the biochemical activities of three M. xanthus RtcB paralogs. We found that M. xanthus RtcB1 resembles E. coli RtcB in its ability to perform intra- and intermolecular sealing of a HORNAp substrate and capping of a DNA 3'-PO4 end. M. xanthus RtcB2 can splice HORNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast, M. xanthus RtcB3 is distinctively feeble at ligating the HORNAp substrate, although it readily caps a DNA 3'-PO4 end. The novelty of M. xanthus RtcB3 is its capacity to cap DNA and RNA 5'-PO4 ends to form GppDNA and GppRNA products, respectively. As such, RtcB3 joins a growing list of enzymes (including RNA 3'-phosphate cyclase RtcA and thermophilic ATP-dependent RNA ligases) that can cap either end of a polynucleotide substrate. GppDNA formed by RtcB3 can be decapped to pDNA by the DNA repair enzyme aprataxin. RtcB enzymes comprise a widely distributed family of RNA 3'-PO4 ligases distinguished by their formation of 3'-GMP-capped RNAppG and/or DNAppG polynucleotides. The mechanism and biochemical repertoire of E. coli RtcB are well studied, but it is unclear whether its properties apply to the many bacteria that have genes encoding multiple RtcB paralogs. A comparison of the biochemical activities of three M. xanthus paralogs, RtcB1, RtcB2, and RtcB3, shows that not all RtcBs are created equal. The standout findings concern RtcB3, which is (i) inactive as an RNA 3'-PO4 ligase but adept at capping a DNA 3'-PO4 end and (ii) able to cap DNA and RNA 5'-PO4 ends to form GppDNA and GppRNA, respectively. The GppDNA and GppRNA capping reactions are novel nucleic acid modifications.

Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy

The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase [RdRp], polyribonucleotidyl transferase [PRNTase], and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.

MRNA Capping by Venezuelan Equine Encephalitis Virus nsP1: Functional Characterization and Implications for Antiviral Research.

Alphaviruses are known to possess a unique viral mRNA capping mechanism involving the viral nonstructural protein nsP1. This enzyme harbors methyltransferase (MTase) and nsP1 guanylylation (GT) activities catalyzing the transfer of the methyl group from S-adenosylmethionine (AdoMet) to the N7 position of a GTP molecule followed by the formation of an m(7)GMP-nsP1 adduct. Subsequent transfer of m(7)GMP onto the 5' end of the viral mRNA has not been demonstrated in vitro yet. Here we report the biochemical characterization of Venezuelan equine encephalitis virus (VEEV) nsP1. We have developed enzymatic assays uncoupling the different reactions steps catalyzed by nsP1. The MTase and GT reaction activities were followed using a nonhydrolyzable GTP (GIDP) substrate and an original Western blot assay using anti-m3G/m(7)G-cap monoclonal antibody, respectively. The GT reaction is stimulated by S-adenosyl-l-homocysteine (Ado-Hcy), the product of the preceding MTase reaction, and metallic ions. The covalent linking between nsP1 and m(7)GMP involves a phosphamide bond between the nucleotide and a histidine residue. Final guanylyltransfer onto RNA was observed for the first time with an alphavirus nsP1 using a 5'-diphosphate RNA oligonucleotide whose sequence corresponds to the 5' end of the viral genome. Alanine scanning mutagenesis of residues H37, H45, D63, E118, Y285, D354, R365, N369, and N375 revealed their respective roles in MT and GT reactions. Finally, the inhibitory effects of sinefungin, aurintricarboxylic acid (ATA), and ribavirin triphosphate on MTase and capping reactions were investigated, providing possible avenues for antiviral research. Emergence or reemergence of alphaviruses represents a serious health concern, and the elucidation of their replication mechanisms is a prerequisite for the development of specific inhibitors targeting viral enzymes. In particular, alphaviruses are able, through an original reaction sequence, to add to their mRNA a cap required for their protection against cellular nucleases and initiation of viral proteins translation. In this study, the capping of a 5' diphosphate synthetic RNA mimicking the 5' end of an alphavirus mRNA was observed in vitro for the first time. The different steps for this capping are performed by the nonstructural protein 1 (nsP1). Reference compounds known to target the viral capping inhibited nsP1 enzymatic functions, highlighting the value of this enzyme in antiviral development.

Efficient preparation and properties of mRNAs containing a fluorescent cap analog: Anthraniloyl-m(7)GpppG.

A method has been developed for synthesising fluorescently labeled capped mRNA. The method incorporates a single fluorescent molecule as part of the 5'-mRNA or oligonucleotide cap site. The fluorescent molecule, Ant-m(7)GTP is specifically incorporated into the cap site to yield Ant-m(7)GpppG-capped mRNA or oligonucleotide. Efficient capping was observed with 60-100% of the RNA transcripts capped with the fluorescent molecule. The Ant-m(7)G derivative, which has been previously shown to interact with the eukaryotic cap binding protein eIF4E, is shown in this paper to be a substrate for the Vaccinia capping enzyme and the DCP2 decapping enzyme from Arabidopsis. Further, the Ant-m(7)GTP-capped RNA is readily translated. This Ant-m(7)GTP-capped RNA provides an important tool for monitoring capping reactions, translation, and biophysical studies.

Identification of the Active Sites in the Methyltransferases of a Transcribing dsRNA Virus

Many double-stranded RNA (dsRNA) viruses are capable of transcribing and capping RNA within a stable icosahedral viral capsid. The turret of turreted dsRNA viruses belonging to the family Reoviridae is formed by five copies of the turret protein, which contains domains with both 7-N-methyltransferase and 2′-O-methyltransferase activities, and serves to catalyze the methylation reactions during RNA capping. Cypovirus of the family Reoviridae provides a good model system for studying the methylation reactions in dsRNA viruses. Here, we present the structure of a transcribing cypovirus to a resolution of ~3.8Å by cryo-electron microscopy. The binding sites for both S-adenosyl-l-methionine and RNA in the two methyltransferases of the turret were identified. Structural analysis of the turret in complex with RNA revealed a pathway through which the RNA molecule reaches the active sites of the two methyltransferases before it is released into the cytoplasm. The pathway shows that RNA capping reactions occur in the active sites of different turret protein monomers, suggesting that RNA capping requires concerted efforts by at least three turret protein monomers. Thus, the turret structure provides novel insights into the precise mechanisms of RNA methylation.

Regioselective Double Capping of Cyclodextrin Scaffolds

Four different regioselective double capping reactions were applied either to α- or β-cyclodextrin (CD) scaffolds. The first, which relied on the use of a rigid, bulky dialkylating reagent containing two trityl-like subunits, gave access to an A,B,D,E-tetrafunctionalised β-CD regioisomer in large scale reactions. Two further capping reactions, involving the dianions PhP(2-) and S(2- , led to the synthesis of new C(1)-symmetrical β-cyclodextrins in which pairs of neighbouring glucose units are linked by very short spacers. The last double capping reaction described allowed the high-yield preparation of unprecedented α- and β-cyclodextrins containing two sulfate handles. Proximal capping turned out to be favoured for each of the above difunctional reagents. The structural characterisation of the capped species was achieved by thorough NMR investigations as well as by single-crystal X-ray diffraction studies.

Rat molar teeth as a study model for direct pulp capping research in dentistry

The aim of this review is to evaluate the suitability of rat molar teeth in preclinical evaluation of medical devices for direct pulp capping. The ISO standard 7405 states clearly that only non-rodent mammals are suitable species for animal research in dentistry. Furthermore, without clear justification a considerable number of researchers previously rejected results of animal experiments concerning preclinical evaluation of the biocompatibility of dental materials undertaken in rat molar teeth. However, in the past 50 years about 70 studies have been published using rat molar teeth in order to evaluate direct pulp capping, pulpotomies and tissue reactions after pulp exposure. Numerous studies showed that the healing of rat molar pulp tissue after direct pulp capping is histologically comparable with humans and other animal species pulp tissue. Rat molar teeth, including pulp tissue, can be seen anatomically, histologically, biologically, and physiologically as miniature human molar teeth. Hence, the essential biological reactions of the pulp tissue and the interaction during the different stages of wound healing of rat molar teeth are comparable with that of other mammals. Rat molar teeth are a valid study model in order to provide valuable data concerning pulp tissue reaction after direct pulp capping and related questions in dentistry. Therefore, the use of rats may significantly reduce the number of currently used higher animals in research. Tests in higher developed animals should be limited to experiments which clarify inconsistent results. However, some technical difficulties, like the small size of rat molar teeth must be dealt with before undertaking any research.

A Conserved Motif in Region V of the Large Polymerase Proteins of Nonsegmented Negative-Sense RNA Viruses That Is Essential for mRNA Capping

Nonsegmented negative-sense (NNS) RNA viruses cap their mRNA by an unconventional mechanism. Specifically, 5' monophosphate mRNA is transferred to GDP derived from GTP through a reaction that involves a covalent intermediate between the large polymerase protein L and mRNA. This polyribonucleotidyltransferase activity contrasts with all other capping reactions, which are catalyzed by an RNA triphosphatase and guanylyltransferase. In these reactions, a 5' diphosphate mRNA is capped by transfer of GMP via a covalent enzyme-GMP intermediate. RNA guanylyltransferases typically have a KxDG motif in which the lysine forms this covalent intermediate. Consistent with the distinct mechanism of capping employed by NNS RNA viruses, such a motif is absent from L. To determine the residues of L protein required for capping, we reconstituted the capping reaction of the prototype NNS RNA virus, vesicular stomatitis virus, from highly purified components. Using a panel of L proteins with single-amino-acid substitutions to residues universally conserved among NNS RNA virus L proteins, we define a new motif, GxxT[n]HR, present within conserved region V of L protein that is essential for this unconventional mechanism of mRNA cap formation.

Unconventional Mechanism of mRNA Capping by the RNA-Dependent RNA Polymerase of Vesicular Stomatitis Virus

All known eukaryotic and some viral mRNA capping enzymes (CEs) transfer a GMP moiety of GTP to the 5'-diphosphate end of the acceptor RNA via a covalent enzyme-GMP intermediate to generate the cap structure. In striking contrast, the putative CE of vesicular stomatitis virus (VSV), a prototype of nonsegmented negative-strand (NNS) RNA viruses including rabies, measles, and Ebola, incorporates the GDP moiety of GTP into the cap structure of transcribing mRNAs. Here, we report that the RNA-dependent RNA polymerase L protein of VSV catalyzes the capping reaction by an RNA:GDP polyribonucleotidyltransferase activity, in which a 5'-monophosphorylated viral mRNA-start sequence is transferred to GDP generated from GTP via a covalent enzyme-RNA intermediate. Thus, the L proteins of VSV and, by extension, other NNS RNA viruses represent a new class of viral CEs, which have evolved independently from known eukaryotic CEs.

A General RNA-Capping Ribozyme Retains Stereochemistry during Cap Exchange

Numerous natural and artificial ribozymes have been shown to facilitate reactions that invert stereochemistry. Here, we demonstrate that an RNA-capping ribozyme retains stereochemistry at a phosphorus reaction center. The ribozyme synthesizes a broad range of 5'-5' RNA caps by exchanging phosphate groups around the alpha-phosphate found at the 5' terminus of the ribozyme. A ribozyme prepared with an Rp adenosine(5')alpha-thiotetraphosphate cap was found to exchange this cap for an Rp 4-thiouridine(5')alpha-thiotetraphosphate cap when incubated with 4-thiouridine triphosphate. The same Rp capped construct, when incubated with [gamma-(32)P]-ATP, exchanged the unlabeled ATP for a radiolabeled one while maintaining the same stereoconfiguration. In contrast, ribozymes prepared with an Sp cap failed to react even in the presence of thiophilic metal ions such as manganese. The kinetics of capping was also unusual as compared to inverting ribozymes. When the ribozyme was prepared with a triphosphate, capping was found to follow Michaelis-Menten-type kinetics even though the rate of pyrophosphate release was completely independent of nucleotide substrate concentration. Interestingly, the rate of capping and hydrolysis, when summed, was found to be indistinguishable from the rate of pyrophosphate release, indicating that an early rate-limiting step precedes both capping and hydrolysis. Together the retention of stereochemistry and kinetics imply that capping utilizes two inverting chemical steps that are separated by the transient formation of a rate-limiting covalent intermediate. As all protein enzymes that mediate similar capping reactions utilize a covalent intermediate, chemical necessity may have strongly guided the evolution of both protein and RNA-capping catalysts.

Two independently selected capping ribozymes share similar substrate requirements

We report the isolation and characterization of a second capping ribozyme, called 6.17. This ribozyme has substrate requirements that are very similar to a previously isolated capping ribozyme called Iso6. Both ribozymes promote capping and cap exchange reactions with a broad range of nucleotide substrates. The ribozymes mediate a reaction where the terminal phosphate of the nucleotide substrate attacks the alpha-phosphate found at the ribozyme's 5' terminus. This reaction involves the release of pyrophosphate during capping or a nucleotide during cap exchange. The second-order rate constants for 6.17 and Iso6 depend strongly on the length of the phosphate group found on the nucleotide substrate. Nucleoside diphosphates or triphosphates are efficiently utilized, while monophosphates are used approximately 20-fold less efficiently by both ribozymes. These ribozymes also have rates that increase as pH is decreased. Despite these similarities, the ribozymes are not identical and 6.17 performs optimally when incubated with divalent magnesium ions, while Iso6 displays a preference for calcium ions. Further, the ribozymes have globally different secondary structures; 6.17 has a complicated pseudoknot structure consisting of five helical elements, while Iso6 likely consists of four helical elements. We hypothesize that capping proceeds via an invariant phosphate dependent mechanism that imposes a nearly identical "catalytic fingerprint" on these two distinct ribozymes.

Phytochelatin synthase, papain's cousin, in stereo

Supraoptimal levels of essential heavy metals (e.g., Cu and Zn) and trace or higher levels of nonessential heavy metals (e.g., As, Cd, and Hg) pose a threat because they undergo aberrant capping reactions with the thiol groups of proteins, displace endogenous metal cofactors from their cellular binding sites, and promote the formation of active oxygen species (1). One of the most sophisticated mechanisms known to confer protection from metal toxicity is the enzyme-catalyzed synthesis of short-chain peptides, phytochelatins (PCs), which serve as high-affinity, thiol-rich cellular chelators and thereby contribute to the detoxification of heavy metal ions, most notably Cd2+, in vascular plants and some algae, diatoms, fungi, and invertebrates (2). Derived from glutathione (GSH) and related thiols in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by phytochelatin synthases (PC synthases, EC 2.3.2.15) (1, 3, 4), PCs have the general structure (γ-Glu-Cys)n-Xaa, where n = 2–11 and Xaa is usually Gly (Fig. 1). Although studies of PC synthases have largely been concerned with the enzymes from eukaryotes, recent database searches have disclosed PC synthase-like sequences in the genomes of several prokaryotes (1, 5). In and of itself, this finding might not be of particular interest except that all of the prokaryotic PC synthase homologs identified are half the length of their cognates from eukaryotes (220–237 residues compared with 421–506 residues) because they lack the more sequence-variable C-terminal domain characteristic of the latter. Moreover, the one prokaryotic PC synthase homolog to have been assayed for activity, the alr0975 protein from the cyanobacterium Nostoc sp. PCC 7120 (NsPCS), catalyzes the deglycylation of GSH to γ-Glu-Cys at a high rate and the synthesis of PC2 at a relatively low rate (Fig. 1) (5, 6). It is as if the prokaryotic enzymes, despite an overall sequence identity of only 22–36% with the N-terminal domains of the eukaryotic enzymes, consist solely of the core catalytic domain of a canonical eukaryotic PC synthase (Fig. 1) (1, 7, 8). Against this background, the stereo structure of NsPCS in its native and γ-Glu-Cys-acylated state reported by Vivares et al. in a recent issue of PNAS (9) is seminal. The results presented not only represent the first crystal structure for a PC synthase but they also establish, as had been inferred tentatively from kinetic, protein chemical, and site mutagenic analyses of the prototypical eukaryotic PC synthase, Arabidopsis thaliana PC synthase 1 (AtPCS1) (Fig. 1) (1, 8, 10), that these enzymes belong to the papain superfamily and deploy a cysteine protease-like catalytic mechanism.