Peptide Bond Formation Research Articles

Translational impacts of enzymes that modify ribosomal RNA around the peptidyl transferase centre

ABSTRACT Large ribosomal RNAs (rRNAs) are modified heavily post-transcriptionally in functionally important regions but, paradoxically, individual knockouts (KOs) of the modification enzymes have minimal impact on Escherichia coli growth. Furthermore, we recently constructed a strain with combined KOs of five modification enzymes (RluC, RlmKL, RlmN, RlmM and RluE) of the ‘critical region’ of the peptidyl transferase centre (PTC) in 23S rRNA that exhibited only a minor growth defect at 37°C (although major at 20°C). However, our combined KO of modification enzymes RluC and RlmE (not RluE) resulted in conditional lethality (at 20°C). Although the growth rates for both multiple-KO strains were characterized, the molecular explanations for such deficits remain unclear. Here, we pinpoint biochemical defects in these strains. In vitro fast kinetics at 20°C and 37°C with ribosomes purified from both strains revealed, counterintuitively, the slowing of translocation, not peptide bond formation or peptidyl release. Elongation rates of protein synthesis in vivo, as judged by the kinetics of β-galactosidase induction, were also slowed. For the five-KO strain, the biggest deficit at 37°C was in 70S ribosome assembly, as judged by a dominant 50S peak in ribosome sucrose gradient profiles at 5 mM Mg2+. Reconstitution of this 50S subunit from purified five-KO rRNA and ribosomal proteins supported a direct role in ribosome biogenesis of the PTC region modifications per se, rather than of the modification enzymes. These results clarify the importance and roles of the enigmatic rRNA modifications.

The structure of the monobactam-producing thioesterase domain of SulM forms a unique complex with the upstream carrier protein domain

Nonribosomal peptide synthetases (NRPSs) are responsible for the production of important biologically active peptides. The large, multidomain NRPSs operate through an assembly line strategy in which the growing peptide is tethered to carrier domains that deliver the intermediates to neighboring catalytic domains. While most NRPS domains catalyze standard chemistry of amino acid activation, peptide bond formation and product release, some canonical NRPS catalytic domains promote unexpected chemistry. The paradigm monobactam antibiotic sulfazecin is produced through the activity of a terminal thioesterase domain of SulM, which catalyzes an unusual β-lactam forming reaction in which the nitrogen of the C-terminal N-sulfo-2,3-diaminopropionate residue attacks its thioester tether to release the monobactam product. We have determined the structure of the thioesterase domain as both a free-standing domain and a didomain complex with the upstream holo peptidyl-carrier domain. The position of variant lid helices results in an active site pocket that is quite constrained, a feature that is likely necessary to orient the substrate properly for β-lactam formation. Modeling of a sulfazecin tripeptide into the active site identifies a plausible binding mode identifying potential interactions for the sulfamate and the peptide backbone with Arg2849 and Asn2819, respectively. The overall structure is similar to the β-lactone forming thioesterase domain that is responsible for similar ring closure in the production of obafluorin. We further use these insights to enable bioinformatic analysis to identify additional, uncharacterized β-lactam-forming biosynthetic gene clusters by genome mining.

PEARL-Catalyzed Peptide Bond Formation after Chain Reversal by Ureido-Forming Condensation Domains.

A subset of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) are encoded in their biosynthetic gene clusters (BGCs) with enzymes annotated as lantibiotic dehydratases. The functions of these putative lantibiotic dehydratases remain unknown. Here, we characterize an NRPS-PKS BGC with a putative lantibiotic dehydratase from the bacterium Stackebrandtia nassauensis (sna). Heterologous expression revealed several metabolites produced by the BGC, and the omission of selected biosynthetic enzymes revealed the biosynthetic pathway toward these compounds. The final product is a bisarginyl ureidopeptide with an enone electrophile. The putative lantibiotic dehydratase catalyzes peptide bond formation to a Thr that extends the peptide scaffold opposite to the NRPS and PKS biosynthetic direction. The condensation domain of the NRPS SnaA catalyzes the formation of a ureido group, and bioinformatics analysis revealed a distinct active site signature EHHXXHDG of ureido-generating condensation (Curea) domains. This work demonstrates that the annotated lantibiotic dehydratase serves as a separate amide bond-forming machinery in addition to the NRPS, and that the lantibiotic dehydratase enzyme family possesses diverse catalytic activities in the biosynthesis of both ribosomal and nonribosomal natural products.

Reversing the relative time courses of the peptide bond reaction with oligopeptides of different lengths and charged amino acid distributions in the ribosome exit tunnel

The kinetics of the protein elongation cycle by the ribosome depends on intertwined factors. One of these factors is the electrostatic interaction of the nascent protein with the ribosome exit tunnel. In this computational biology theoretical study, we focus on the rate of the peptide bond formation and its dependence on the ribosome exit tunnel electrostatic potential profile. We quantitatively predict how oligopeptides of variable lengths can affect the peptide bond formation rate. We applied the Michaelis-Menten model as previously extended to incorporate the mechano-biochemical effects of forces on the rate of reaction at the catalytic site of the ribosome. For a given pair of carboxy-terminal amino acid substrate at the P- and an aminoacyl-tRNA at the A-sites, the relative time courses of the peptide bond formation reaction can be reversed depending on the oligopeptide sequence embedded in the tunnel and their variable lengths from the P-site. The reversal is predicted to occur from a shift in positions of charged amino acids upstream in the oligopeptidyl-tRNA at the P-site. The position shift must be adjusted by clever design of the oligopeptide probes using the electrostatic potential profile along the exit tunnel axial path. These predicted quantitative results bring strong evidence of the importance and relative contribution of the electrostatic interaction of the ribosome exit tunnel with the nascent peptide chain during elongation.

Rapid Synthesis of Peptides Mediated by PPh3−I2

AbstractWe report a mild, operationally simple, convenient, and rapid method of peptide bond formation using triphenylphosphine and iodine. The developed protocol was utilized to couple coded, non‐coded, and challenging amino acids in 64–92 % yield. Sterically hindered α,α‐disubstituted amino acids, which are important constituents of natural peptides, were also coupled in good yield. Mild reaction conditions in open air produce peptide bonds in a short time (30 min) without racemization.

Competing Reaction Mechanisms of Peptide Bond Formation in Water Revealed by Deep Potential Molecular Dynamics and Path Sampling.

The formation of an amide bond is an essential step in the synthesis of materials and drugs, and in the assembly of amino acids to form peptides. The mechanism of this reaction has been studied extensively, in particular to understand how it can be catalyzed, but a representation capable of explaining all the experimental data is still lacking. Numerical simulation should provide the necessary molecular description, but the solvent involvement poses a number of challenges. Here, we combine the efficiency and accuracy of neural network potential-based reactive molecular dynamics with the extensive and unbiased exploration of reaction pathways provided by transition path sampling. Using microsecond-scale simulations at the density functional theory level, we show that this method reveals the presence of two competing distinct mechanisms for peptide bond formation between alanine esters in aqueous solution. We describe how both reaction pathways, via a general base catalysis mechanism and via direct cleavage of the tetrahedral intermediate respectively, change with pH. This result contrasts with the conventional mechanism involving a single pathway in which only the barrier heights are affected by pH. We show that this new proposal involving two competing mechanisms is consistent with the experimental data, and we discuss the implications for peptide bond formation under prebiotic conditions and in the ribosome. Our work shows that integrating deep potential molecular dynamics with path sampling provides a powerful approach for exploring complex chemical mechanisms.

On the Re-Creation of Protoribosome Analogues in the Lab.

The evolution of the translation system is a fundamental issue in the quest for the origin of life. A feasible evolutionary scenario necessitates the autonomous emergence of a protoribosome capable of catalyzing the synthesis of the initial peptides. The peptidyl transferase center (PTC) region in the modern ribosomal large subunit is believed to retain a vestige of such a prebiotic non-coded protoribosome, which would have self-assembled from random RNA chains, catalyzed peptide bond formation between arbitrary amino acids, and produced short peptides. Recently, three research groups experimentally demonstrated that several distinct dimeric constructs of protoribosome analogues, derived predicated on the approximate 2-fold rotational symmetry inherent in the PTC region, possess the ability to spontaneously fold, dimerize, and catalyze the formation of peptide bonds and of short peptides. These dimers are examined, aiming at retrieving information concerned with the characteristics of a prebiotic protoribosome. The analysis suggests preconditions for the laboratory re-creation of credible protoribosome analogues, including the preference of a heterodimer protoribosome, contradicting the common belief in the precedence of homodimers. Additionally, it derives a dynamic process which possibly played a role in the spontaneous production of the first bio-catalyzed peptides in the prebiotic world.

Biodegradation Study of Styrene-Butadiene Composites with Incorporated Arthrospira platensis Biomass.

The preparation of polymer composites that incorporate material of a biogenic nature in the polymer matrices may lead to a reduction in fossil polymer consumption and a potentially higher biodegradability. Furthermore, microalgae biomass as biogenic filler has the advantage of fast growth and high tolerance to different types of culture media with higher production yields than those provided by the biomass of terrestrial crops. On the other hand, algal biomass can be a secondary product in wastewater treatment processes. For the present study, an SBS polymer composite (SBSC) containing 25% (w/w) copolymer SBS1 (linear copolymer: 30% styrene and 70% butadiene), 50% (w/w) copolymer SBS2 (linear copolymer: 40% styrene and 60% butadiene), and 25% (w/w) paraffin oil was prepared. Arthrospira platensis biomass (moisture content 6.0 ± 0.5%) was incorporated into the SBSC in 5, 10, 20, and 30% (w/w) ratios to obtain polymer composites with spirulina biomass. For the biodegradation studies, the ISO 14855-1:2012(E) standard was applied, with slight changes, as per the specificity of our experiments. The degradation of the studied materials was followed by quantitatively monitoring the CO2 resulting from the degradation process and captured by absorption in NaOH solution 0.5 mol/L. The structural and morphological changes induced by the industrial composting test on the materials were followed by physical-mechanical, FTIR, SEM, and DSC analysis. The obtained results were compared to create a picture of the material transformation during the composting period. Thus, the collected data indicate two biodegradation processes, of the polymer and the biomass, which take place at the same time at different rates, which influence each other. On the other hand, it is found that the material becomes less ordered, with a sponge-like morphology; the increase in the percentage of biomass leads to an advanced degree of degradation of the material. The FTIR analysis data suggest the possibility of the formation of peptide bonds between the aromatic nuclei in the styrene block and the molecular residues resulting from biomass biodegradation. It seems that in industrial composting conditions, the area of the polystyrene blocks from the SBS-based composite is preferentially transformed in the process.

C-C Bond Cleavage-Induced C- to N-Acyl Transfer for Synthesis of Amides.

A new approach for the preparation of amides was developed using C-C bond cleavage that initiates C- to N-acyl transfer, employing activated ketones as acylation reagents and amine nucleophiles. The reaction was operational under the coupling reagent system that is commonly utilized for peptide bond formations. The method enables practical preparation of amides using linear and cyclic ketone substrates under mild conditions.

Highly defective ultra-small tetravalent MOF nanocrystals

The size and defects in crystalline inorganic materials are of importance in many applications, particularly catalysis, as it often results in enhanced/emerging properties. So far, applying the strategy of modulation chemistry has been unable to afford high-quality functional Metal–Organic Frameworks (MOFs) nanocrystals with minimized size while exhibiting maximized defects. We report here a general sustainable strategy for the design of highly defective and ultra-small tetravalent MOFs (Zr, Hf) crystals (ca. 35% missing linker, 4–6 nm). Advanced characterizations have been performed to shed light on the main factors governing the crystallization mechanism and to identify the nature of the defects. The ultra-small nanoMOFs showed exceptional performance in peptide hydrolysis reaction, including high reactivity, selectivity, diffusion, stability, and show emerging tailorable reactivity and selectivity towards peptide bond formation simply by changing the reaction solvent. Therefore, these highly defective ultra-small M(IV)-MOFs particles open new perspectives for the development of heterogeneous MOF catalysts with dual functions.

RNA-Templated Peptide Bond Formation Promotes L-Homochirality.

The world in which we live is homochiral. The ribose units that form the backbone of DNA and RNA are all D-configured and the encoded amino acids that comprise the proteins of all living species feature an all-L-configuration at the α-carbon atoms. The homochirality of α-amino acids is essential for folding of the peptides into well-defined and functional 3D structures and the homochirality of D-ribose is crucial for helix formation and base-pairing. The question of why nature uses only encoded L-α-amino acids is not understood. Herein, we show that an RNA-peptide world, in which peptides grow on RNAs constructed from D-ribose, leads to the self-selection of homo-L-peptides, which provides a possible explanation for the homo-D-ribose and homo-L-amino acid combination seen in nature.

The SecM arrest peptide traps a pre-peptide bond formation state of the ribosome

Nascent polypeptide chains can induce translational stalling to regulate gene expression. This is exemplified by the E. coli secretion monitor (SecM) arrest peptide that induces translational stalling to regulate expression of the downstream encoded SecA, an ATPase that co-operates with the SecYEG translocon to facilitate insertion of proteins into or through the cytoplasmic membrane. Here we present the structure of a ribosome stalled during translation of the full-length E. coli SecM arrest peptide at 2.0 Å resolution. The structure reveals that SecM arrests translation by stabilizing the Pro-tRNA in the A-site, but in a manner that prevents peptide bond formation with the SecM-peptidyl-tRNA in the P-site. By employing molecular dynamic simulations, we also provide insight into how a pulling force on the SecM nascent chain can relieve the SecM-mediated translation arrest. Collectively, the mechanisms determined here for SecM arrest and relief are also likely to be applicable for a variety of other arrest peptides that regulate components of the protein localization machinery identified across a wide range of bacteria lineages.

RAPP-containing arrest peptides induce translational stalling by short circuiting the ribosomal peptidyltransferase activity

Arrest peptides containing RAPP (ArgAlaProPro) motifs have been discovered in both Gram-positive and Gram-negative bacteria, where they are thought to regulate expression of important protein localization machinery components. Here we determine cryo-EM structures of ribosomes stalled on RAPP arrest motifs in both Bacillus subtilis and Escherichia coli. Together with molecular dynamics simulations, our structures reveal that the RAPP motifs allow full accommodation of the A-site tRNA, but prevent the subsequent peptide bond from forming. Our data support a model where the RAP in the P-site interacts and stabilizes a single hydrogen atom on the Pro-tRNA in the A-site, thereby preventing an optimal geometry for the nucleophilic attack required for peptide bond formation to occur. This mechanism to short circuit the ribosomal peptidyltransferase activity is likely to operate for the majority of other RAPP-like arrest peptides found across diverse bacterial phylogenies.

Decomposing bulk signals to reveal hidden information in processive enzyme reactions: A case study in mRNA translation.

Processive enzymes like polymerases or ribosomes are often studied in bulk experiments by monitoring time-dependent signals, such as fluorescence time traces. However, due to biomolecular process stochasticity, ensemble signals may lack the distinct features of single-molecule signals. Here, we demonstrate that, under certain conditions, bulk signals from processive reactions can be decomposed to unveil hidden information about individual reaction steps. Using mRNA translation as a case study, we show that decomposing a noisy ensemble signal generated by the translation of mRNAs with more than a few codons is an ill-posed problem, addressable through Tikhonov regularization. We apply our method to the fluorescence signatures of in-vitro translated LepB mRNA and determine codon-position dependent translation rates and corresponding state-specific fluorescence intensities. We find a significant change in fluorescence intensity after the fourth and the fifth peptide bond formation, and show that both codon position and encoded amino acid have an effect on the elongation rate. This demonstrates that our approach enhances the information content extracted from bulk experiments, thereby expanding the range of these time- and cost-efficient methods.

Boronic Acid-Catalyzed Peptide Bond Formation from Serine- and Threonine-Derived Inert Esters

Boronic Acid-Catalyzed Peptide Bond Formation from Serine- and Threonine-Derived Inert Esters

Hydroxy-directed peptide bond formation from α-amino acid-derived inert esters enabled by boronic acid catalysis.

A boronic acid-catalyzed peptide bond formation from α-amino acid methyl esters is described. The catalysis showed high chemoselectivity for β-hydroxy-α-amino esters, affording the peptides in high to excellent yields with high functional group tolerance. This hydroxy-directed peptide bond formation could be applicable to oligopeptide syntheses. This is the first successful example of organoboron-catalyzed peptide bond formation from α-amino acid-derived inert esters.

TFPN-mediated racemization/epimerization-free amide and peptide bond formation

A new electron-deficient aromatic-based coupling reagent, tetrafluorophthalonitrile (TFPN), designed according to the concept of dual coupling reagents, is developed to facilitate amide and peptide bond formation in a one-pot, two-step manner.

DIMT1 and Ribosomal Function: How Ribosomal Biogenesis Influences Lifespan and Energy Homeostasis

Ribosomes are essential molecular machines responsible for synthesizing proteins, a process vital for cellular growth, repair, and overall homeostasis. Central to ribosomal function is ribosomal RNA (rRNA), which not only provides the structural framework of the ribosome but also catalyzes peptide bond formation during translation. Recent research has highlighted the significant role of ribosomal biogenesis in cellular aging and disease, emphasizing the impact of abnormalities in ribosome production and function. This review focuses on the complicated mechanisms underlying ribosome assembly, with particular emphasis on the role of rRNA and the novel involvement of the dimethyladenosine transferase 1 (DIMT1) gene. DIMT1 is responsible for specific methylation modifications of rRNA during ribosome maturation, contributing to the proper structure and functionality of ribosomes. Additionally, it is involved in regulating mitochondrial function, which is the cell’s energy generation core. Recent evidence suggests that DIMT1 may play a crucial part in aging and longevity by increasing mitochondrial efficiency and altering cellular responses to metabolic stress. The gene’s conservation across species highlights its potential as a therapeutic target for interventions to delay the development of age-related diseases and extend a healthy lifespan.

Synergistic Photo-Redox Catalysis for Peptide Bond Formation

Synergistic Photo-Redox Catalysis for Peptide Bond Formation

A novel MB-tagged aptasensor for Aflatoxin B1 detection in food using Fe3O4 nanoparticles substantiated with in silico modelling

Aspergillus fungi species found in wheat, maize, rice, and other agricultural products produce the carcinogenic mycotoxin known as Aflatoxin B1 (AFB1), which can cause cancer in animals and humans. Consequently, recent interest has surged regarding the need for inexpensive, selective, and sensitive sensors to detect AFB1 in food. An ultrasensitive electrochemical aptasensor for AFB1 analysis was constructed using carboxylated multiwalled carbon nanotubes (cMWCNTs) and iron oxide (Fe3O4) nanoparticles (NP) on a glassy carbon electrode (GCE). The peptide bond formation by EDC coupling between the aptamer and cMWCNTs-Fe3O4 NP composite exhibited a strong anodic redox response from AFB1 using cyclic voltammetry (CV) in this study. Applying differential pulse voltammetry (DPV), the GCE/cMWCNTs-Fe3O4 NP aptasensor exhibited very low limits of detection (LOD) and quantification (LOQ) of 0.43 fg mL−1 and 1.44 fg mL−1 respectively over a calibration ranging from 0.50 fg mL−1 to 5.00 fg mL−1. For actual sample analysis, excellent spike recoveries from 95 to 105% were obtained for corn and rice flour. Single particle ICP-MS (spICP-MS) confirmed the average mass-based diameter of the synthesized Fe3O4 NPs to be in the nano-range (d ≈ 20 nm), the properties of which are essential for the facilitation of strong electron transfer in DPV sensing. Finally, density functional theory and molecular docking studies predicted the sensing mechanism and supported deductions based on the AFB1 capture by the employed aptamer respectively. As part of South Africa’s quality control and regulatory frameworks, this study aims to contribute toward the prevention of AFB1 exposure in foods.