Fundamentals behind the specificity of Cysteinyl-tRNA synthetase: MD and QM/MM joint investigations.

Cysteinyl-tRNA synthetase (CysRS) catalyzes the aminoacylation reaction of cysteine to its cognate tRNACys in the first step of protein translation. It is found that CysRS is different from other aaRSs as it transfers cysteine without the need for an editing reaction, which is not applicable in the case of serine despite the similarity in their structures. Surprisingly, the reasons why CysRS has high amino acid specificity are not clear yet. In this research, the binding configurations of Cys-AMP and its near-cognate amino acid Ser-AMP with CysRS are compared by Molecular Dynamics (MD). The results reveal that CysRS screens the substrate Cys-AMP to a certain extent in the process of combination and recognition, thus providing a guarantee for the high selectivity of the next reaction. While Ser-AMP is in a folded state in CysRS. In the meanwhile, the interaction between Cys-AMP and Zn963 in CysRS is much stronger than Ser-AMP. The substrate-assisted aminoacylation mechanism in CysRS is also explored by Quantum Mechanics/Molecular Mechanics (QM/MM) modeling. According to the QM/MM potential energies, the energy barrier of TSCys-AMP is 91.75 kJ/mol, while that of TSSer-AMP is close to 150 kJ/mol. Based on thermochemistry calculations, it is found that the product of Cys-AMP is more stable than the reactant. In contrast, Ser-AMP has a reactant that is more stable than its product. As a result, it reflects that the specificity of CysRS originates from both the kinetic and thermodynamical perspectives of the reaction. Our investigations demonstrate comprehensively on how CysRS recognizes and catalyzes the substrate Cys-AMP, hoping to provide some guidance for researchers in this area.

Applications and Potential of In Silico Approaches for Psychedelic Chemistry.

Molecular-level investigations of the Central Nervous System have been revolutionized by the development of computational methods, computing power, and capacity advances. These techniques have enabled researchers to analyze large amounts of data from various sources, including genomics, in vivo, and in vitro drug tests. In this review, we explore how computational methods and informatics have contributed to our understanding of mental health disorders and the development of novel drugs for neurological diseases, with a special focus on the emerging field of psychedelics. In addition, the use of state-of-the-art computational methods to predict the potential of drug compounds and bioinformatic tools to integrate disparate data sources to create predictive models is also discussed. Furthermore, the challenges associated with these methods, such as the need for large datasets and the diversity of in vitro data, are explored. Overall, this review highlights the immense potential of computational methods and informatics in Central Nervous System research and underscores the need for continued development and refinement of these techniques and more inclusion of Quantitative Structure-Activity Relationships (QSARs).

The Hydrolysis Rate of Paraoxonase-1 Q and R Isoenzymes: An In Silico Study Based on In Vitro Data.

Human serum paraoxonase-1 (PON1) is an important hydrolase-type enzyme found in numerous tissues. Notably, it can exist in two isozyme-forms, Q and R, that exhibit different activities. This study presents an in silico (QSAR, Docking, MD and QM/MM) study of a set of compounds on the activity towards the PON1 isoenzymes (QPON1 and RPON1). Different rates of reaction for the Q and R isoenzymes were analyzed by modelling the effect of Q192R mutation on active sites. It was concluded that the Q192R mutation is not even close to the active site, while it is still changing the geometry of it. Using the combined genetic algorithm with multiple linear regression (GA-MLR) technique, several QSAR models were developed and relative activity rates of the isozymes of PON1 explained. From these, two QSAR models were selected, one each for the QPON1 and RPON1. Best selected models are four-variable MLR models for both Q and R isozymes with squared correlation coefficient R2 values of 0.87 and 0.83, respectively. In addition, the applicability domain of the models was analyzed based on the Williams plot. The results were discussed in the light of the main factors that influence the hydrolysis activity of the PON1 isozymes.

Antagonizing binding of cell cycle and apoptosis regulatory protein 1 (CARP-1) to the NEMO/IKKγ protein enhances the anticancer effect of chemotherapy.

NF-κB is a pro-inflammatory transcription factor that critically regulates immune responses and other distinct cellular pathways. However, many NF-κB-mediated pathways for cell survival and apoptosis signaling in cancer remain to be elucidated. Cell cycle and apoptosis regulatory protein 1 (CARP-1 or CCAR1) is a perinuclear phosphoprotein that regulates signaling induced by anticancer chemotherapy and growth factors. Although previous studies have reported that CARP-1 is a part of the NF-κB proteome, regulation of NF-κB signaling by CARP-1 and the molecular mechanism(s) involved are unclear. Here, we report that CARP-1 directly binds the NF-κB-activating kinase IκB kinase subunit γ (NEMO or NF-κB essential modulator) and regulates the chemotherapy-activated canonical NF-κB pathway. Importantly, blockade of NEMO-CARP-1 binding diminished NF-κB activation, indicated by reduced phosphorylation of its subunit p65/RelA by the chemotherapeutic agent adriamycin (ADR), but not NF-κB activation induced by tumor necrosis factor α (TNFα), interleukin (IL)-1ß, or epidermal growth factor. High-throughput screening of a chemical library yielded a small molecule inhibitor of NEMO-CARP-1 binding, termed selective NF-κB inhibitor 1 (SNI)-1). We noted that SNI-1 enhances chemotherapy-dependent growth inhibition of a variety of cancer cells, including human triple-negative breast cancer (TNBC) and patient-derived TNBC cells in vitro, and attenuates chemotherapy-induced secretion of the pro-inflammatory cytokines TNFα, IL-1ß, and IL-8. SNI-1 also enhanced ADR or cisplatin inhibition of murine TNBC tumors in vivo and reduced systemic levels of pro-inflammatory cytokines. We conclude that inhibition of NEMO-CARP-1 binding enhances responses of cancer cells to chemotherapy.

Hydrolysis Mechanism of the Linkers by Matrix Metalloproteinase-9 Using QM/MM Calculations.

Activatable cell-penetrating peptides (ACPPs) are known to be able to decrease the cytotoxicity of cell-penetrating peptide (CPP)-based drug delivery systems. Furthermore, they can improve the targeting of CPPs when specifically recognized and hydrolyzed by characteristic proteases. A comprehensive and profound understanding of the recognition and hydrolysis process will provide a better design of the ACPP-based drug delivery system. Previous studies have clearly described how ACPPs are recognized and bound by MMPs. However, the hydrolysis mechanism of ACPPs is still unsolved. This work focuses on a proteinase-sensitive cleavable linker of ACPPs (PLGLAG), the key structure for recognition and hydrolysis, trying to determine the mechanism by which MMP-9 hydrolyzes its substrate PLGLAG. The quantum mechanics/molecular mechanics (QM/MM) calculations herein show that MMP-9 proteolysis is a water-mediated four-step reaction. More specifically, it consists of (i) nucleophilic attack, (ii) hydrogen-bond rearrangement, (iii) proton transfer, and finally (iv) amide bond rupture. Considering the reversibility of multistep reaction, the second step (i.e., hydrogen-bond rearrangement) has the highest barrier and is the rate-limiting step in the hydrolysis of PLGLAG. The possible design and improvement of the key P1 and P1' sites are also explored through mutations. The present results indicate that, while the mutations affect the reaction energy barriers and the rate-limiting steps, all mutants considered could be hydrolyzed by MMP-9. To provide further insights, the hydrolysis mechanism of MMP-2, which has a similar hydrolysis process to that of MMP-9 but with different reaction barriers, is also studied and compared. As a result, this work provides detailed insights into the hydrolysis mechanism of ACPPs by MMP-9 and, thus, also possible insights for the development of new strategies for ACPP-based delivery systems.

Molecular Dynamics Simulations of a Cytochrome P450 from Tepidiphilus thermophilus (P450-TT) Reveal How Its Substrate-Binding Channel Opens.

Cytochrome P450s (P450) are important enzymes in biology with useful biochemical reactions in, for instance, drug and xenobiotics metabolisms, biotechnology, and health. Recently, the crystal structure of a new member of the CYP116B family has been resolved. This enzyme is a cytochrome P450 (CYP116B46) from Tepidiphilus thermophilus (P450-TT) and has potential for the oxy-functionalization of organic molecules such as fatty acids, terpenes, steroids, and statins. However, it was thought that the opening to its hitherto identified substrate channel was too small to allow organic molecules to enter. To investigate this, we performed molecular dynamics simulations on the enzyme. The results suggest that the crystal structure is not relaxed, possibly due to crystal packing effects, and that its tunnel structure is constrained. In addition, the simulations revealed two key amino acid residues at the mouth of the channel; a glutamyl and an arginyl. The glutamyl's side chain tightens and relaxes the opening to the channel in conjunction with the arginyl's, though the latter's side chain is less dramatically changed after the initial relaxation of its conformations. Additionally, it was observed that the effect of increased temperature did not considerably affect the dynamics of the enzyme fold, including the relative solvent accessibility of the amino acid residues that make up the substrate channel wall even as compared to the changes that occurred at room temperature. Interestingly, the substrate channel became distinguishable as a prominent tunnel that is likely to accommodate small- to medium-sized organic molecules for bioconversions. That is, P450-TT has the ability to pass appropriate organic substrates to its active site through its elaborate substrate channel, and notably, is able to control or gate any molecules at the opening to this channel.

The K2(9-ethylguanine)122+ quadruplex is more stable to unimolecular dissociation than the K(9-ethylguanine)8+ quadruplex in the gas phase: a BIRD, energy resolved SORI-CID, IRMPD spectroscopic, and computational study.

A combination of experimental trapped-ion mass spectrometric studies and computational chemistry has been used in the present work to assess the intrinsic properties of the potassiated 9-ethylguanine (9eG) self-assembled quadruplex, K2(9eG)122+, in the gas phase. Infrared multiple photon dissociation (IRMPD) spectroscopy in the N-H/C-H stretching region (2700-3800 cm-1) revealed that this G-quadruplex is a sandwich-type structure with two G-tetrads sandwiching each of the two K+, very similar to the structure determined previously for the K(9eG)8+ complexes. The stability of K2(9eG)122+ toward unimolecular dissociation and its binding energy were examined using energy-resolved sustained off-resonance collision induced dissociation (SORI-CID) and blackbody infrared radiative dissociation (BIRD) kinetics experiments. SORI-CID experiments showed that the self-assembled K2(9eG)122+ complex undergoes charge separation forming K(9eG)8+ and K(9eG)4+ compared to K(9eG)8+ which loses neutral 9eG. More interestingly, K2(9eG)122+ is more stable toward unimolecular dissociation activated by SORI-CID than the K(9eG)8+ complex. Temperature dependent BIRD kinetics for K2(9eG)122+ were consistent with energy-resolved SORI-CID results showing K2(9eG)122+ to have an activation energy of 225 ± 15 kJ mol-1, approximately 50 kJ mol-1 greater than that determined for K(9eG)8+. The extra stability of K2(9eG)122+ is apparently not thermodynamic stability, but most likely due to an energy barrier for dissociation.

Multiscale Computational Study on the Catalytic Mechanism of the Nonmetallo Amidase Maleamate Amidohydrolase (NicF).

Maleamate amidohydrolase (NicF) is a key enzyme in vitamin B3 metabolism that catalyzes the hydrolysis of maleamate to produce maleic acid and ammonia. Unlike most members from the amidohydrolase superfamily it does not require a metal ion. Here, we use multiscale computational enzymology to investigate the catalytic mechanism, substrate binding, oxyanion hole, and roles of key active site residues of NicF from Bordetella bronchiseptica. In particular, molecular dynamics (MD) simulations, quantum mechanics/molecular mechanics (QM/MM) and QTAIM methods have been applied. The mechanism of the NicF-catalyzed reaction proceeds by a nucleophilic addition-elimination sequence involving the formation of a thioester enzyme intermediate (IC2 in stage 1) followed by hydrolysis of the thioester bond to form the products (stage 2). Consequently, the formation of IC2 in stage 1 is the rate-limiting step with a barrier of 88.8 kJ·mol-1 relative to the reactant complex, RC. Comparisons with related metal-dependent enzymes, particularly the zinc-dependent nicotinamidase from Streptococcus pneumonia (SpNic), have also been made to further illustrate unique features of the present mechanism. Along with -NH- donor groups of the oxyanion hole (i.e., HN-Thr146, HN-Cys150), the active site ß-hydroxyl of threonine (HO-ßThr146) is concluded to play a role in stabilizing the carbonyl oxygen of maleamate during the mechanism.

We've Come a Long Way, Baby: Announcing a Special Issue to Commemorate the Publication of Molecule's 20,000th Paper.

On behalf of my Section Editor-in-Chief co-author colleagues I am pleased to announce a Special Issue to commemorate the recent publication of Molecules' 20,000th paper [...].

An Assessment of Computational Methods for Calculating Accurate Structures and Energies of Bio-Relevant Polysulfur/Selenium-Containing Compounds.

The heavier chalcogens sulfur and selenium are important in organic and inorganic chemistry, and the role of such chalcogens in biological systems has recently gained more attention. Sulfur and, to a lesser extent selenium, are involved in diverse reactions from redox signaling to antioxidant activity and are considered essential nutrients. We investigated the ability of the DFT functionals (B3LYP, B3PW91, ωB97XD, M06-2X, and M08-HX) relative to electron correlation methods MP2 and QCISD to produce reliable and accurate structures as well as thermochemical data for sulfur/selenium-containing systems. Bond lengths, proton affinities (PA), gas phase basicities (GPB), chalcogen⁻chalcogen bond dissociation enthalpies (BDE), and the hydrogen affinities (HA) of thiyl/selenyl radicals were evaluated for a range of small polysulfur/selenium compounds and cysteine per/polysulfide. The S⁻S bond length was found to be the most sensitive to basis set choice, while the geometry of selenium-containing compounds was less sensitive to basis set. In mixed chalcogens species of sulfur and selenium, the location of the sulfur atom affects the S⁻Se bond length as it can hold more negative charge. PA, GPB, BDE, and HA of selenium systems were all lower, indicating more acidity and more stability of radicals. Extending the sulfur chain in cysteine results in a decrease of BDE and HA, but these plateau at a certain point (199 kJ mol-1 and 295 kJ mol-1), and PA and GPB are also decreased relative to the thiol, indicating that the polysulfur species exist as thiolates in a biological system. In general, it was found that ωB97XD/6-311G(2d,p) gave the most reasonable structures and thermochemistry relative to benchmark calculations. However, nuances in performance are observed and discussed.

A water-mediated and substrate-assisted aminoacylation mechanism in the discriminating aminoacyl-tRNA synthetase GlnRS and non-discriminating GluRS.

Glutaminyl-tRNA synthetase (GlnRS) catalyzes the aminoacylation of glutamine to the corresponding tRNAGln. However, most bacteria and all archaea lack GlnRS and thus an indirect noncanonical aminoacylation is required. With the assistance of a non-discriminating version of Glutamyl-tRNA synthetases (ND-GluRS) the tRNAGln is misaminoacylated by glutamate. In this study, we have computationally investigated the aminoacylation mechanism in GlnRS and ND-GluRS employing Molecular Dynamics (MD) simulations, Quantum Mechanics (QM) cluster and Quantum Mechanics/Molecular Mechanics (QM/MM) calculations. Our investigations demonstrated the feasibility of a water-mediated, substrate-assisted catalysis pathway with rate limiting steps occurring at energy barriers of 25.0 and 25.4 kcal mol-1 for GlnRS and ND-GluRS, respectively. A conserved lysine residue participates in a second proton transfer to facilitate the departure of the adenosine monophosphate (AMP) group. Thermodynamically stable (-29.9 and -9.3 kcal mol-1 for GlnRS and ND-GluRS) product complexes are obtained only when the AMP group is neutral.

Substrate-Assisted and Enzymatic Pretransfer Editing of Nonstandard Amino Acids by Methionyl-tRNA Synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are central to a number of physiological processes, including protein biosynthesis. In particular, they activate and then transfer their corresponding amino acid to the cognate tRNA. This is achieved with a generally remarkably high fidelity by editing against incorrect standard and nonstandard amino acids. Using docking, molecular dynamics (MD), and hybrid quantum mechanical/molecular mechanics methods, we have investigated mechanisms by which methionyl-tRNA synthetase (MetRS) may edit against the highly toxic, noncognate, amino acids homocysteine (Hcy) and its oxygen analogue, homoserine (Hse). Substrate-assisted editing of Hcy-AMP in which its own phosphate acts as the mechanistic base occurs with a rate-limiting barrier of 98.2 kJ mol(-1). This step corresponds to nucleophilic attack of the Hcy side-chain sulfur at its own carbonyl carbon (CCarb). In contrast, a new possible editing mechanism is identified in which an active site aspartate (Asp259) acts as the base. The rate-limiting step is now rotation about the substrate's aminoacyl Cß-Cγ bond with a barrier of 27.5 kJ mol(-1), while for Hse-AMP, the rate-limiting step is cleavage of the CCarb-OP bond with a barrier of 30.9 kJ mol(-1). A similarly positioned aspartate or glutamate also occurs in the homologous enzymes LeuRS, IleRS, and ValRS, which also discriminate against Hcy. Docking and MD studies suggest that at least in the case of LeuRS and ValRS, a similar editing mechanism may be possible.

Computational investigations on the catalytic mechanism of maleate isomerase: the role of the active site cysteine residues.

The maleate isomerase (MI) catalysed isomerization of maleate to fumarate has been investigated using a wide range of computational modelling techniques, including small model DFT calculations, QM-cluster approach, quantum mechanical/molecular mechanical approach (QM/MM in the ONIOM formalism) and molecular dynamics simulations. Several fundamental questions regarding the mechanism were answered in detail, such as the activation and stabilization of the catalytic Cys in a rather hydrophobic active site. The two previously proposed mechanisms were considered, where either enediolate or succinyl-Cys intermediate forms. Small model calculations as well as an ONIOM-based approach suggest that an enediolate intermediate is too unstable. Furthermore, the formation of succinyl-Cys intermediate via the nucleophilic attack of Cys76(-) on the substrate C2 (as proposed experimentally) was found to be energetically unfeasible in both QM-cluster and ONIOM approaches. Instead, our results show that Cys194, upon activation via the substrate, acts as a nucleophile and Cys76 acts as an acid/base catalyst, forming a succinyl-Cys intermediate in a concerted fashion. Indeed, the calculated PA of Cys76 is always higher than that of Cys194 before or upon substrate binding in the active site. Furthermore, the mechanism proceeds via multiple steps by substrate rotation around C2-C3 with the assistance of the now negatively charged Cys76, leading to the formation of fumarate. Finally, our calculated barrier is in good agreement with experiment. These findings represent a novel mechanism in the racemase superfamily.

The catalytic formation of leukotriene C4: a critical step in inflammatory processes.

Leukotrienes (LT) are a family of drug-like molecules involved in the pathobiology of bronchial asthma and are responsible for smooth muscle contraction. Leukotriene C4 synthase (LTC4S) is a nuclear-membrane enzyme responsible for the conjugation of leukotriene A4 (LTA4) to glutathione to form LTC4, a cysteinyl leukotriene. In this study, the mechanism of LTA4 binding by LTC4S has been computationally examined. More specifically, docking and molecular dynamics simulations were used to gain insight into the substrate-bound active site. These studies identified two possible orientations for bound LTA4: 'tail-to-head' and 'head-to-tail'. An ONIOM(QM/MM) approach was then used to elucidate the mechanism by which glutathione may add to LTA4. In particular, the thiolate of glutathione acts as a nucleophile attacking C6 of LTA4 forming a S-C6 bond. Concomitantly, a proton is transferred from the guanidinium of Arg31 to the epoxide ring oxygen. This results in opening of the epoxide ring and stabilization of the LTC4 product complex. Within the present computational methodology the 'tail-to-head' orientation appears to be the most likely substrate orientation.

A multi-scale computational study on the mechanism of Streptococcus pneumoniae Nicotinamidase (SpNic).

Nicotinamidase (Nic) is a key zinc-dependent enzyme in NAD metabolism that catalyzes the hydrolysis of nicotinamide to give nicotinic acid. A multi-scale computational approach has been used to investigate the catalytic mechanism, substrate binding and roles of active site residues of Nic from Streptococcus pneumoniae (SpNic). In particular, density functional theory (DFT), molecular dynamics (MD) and ONIOM quantum mechanics/molecular mechanics (QM/MM) methods have been employed. The overall mechanism occurs in two stages: (i) formation of a thioester enzyme-intermediate (IC2) and (ii) hydrolysis of the thioester bond to give the products. The polar protein environment has a significant effect in stabilizing reaction intermediates and in particular transition states. As a result, both stages effectively occur in one step with Stage 1, formation of IC2, being rate limiting barrier with a cost of 53.5 kJ·mol-1 with respect to the reactant complex, RC. The effects of dispersion interactions on the overall mechanism were also considered but were generally calculated to have less significant effects with the overall mechanism being unchanged. In addition, the active site lysyl (Lys103) is concluded to likely play a role in stabilizing the thiolate of Cys136 during the reaction.

Sulfilimine bond formation in collagen IV.

The collagen IV network plays a crucial role in providing structural support and mechanical integrity to the basement membrane and surrounding tissues. A key aspect of this network is the formation of intra- and inter-collagen fibril crosslinks. One particular crosslink, an inter-residue sulfilimine bond, has been found, so far, to be unique to collagen IV. More specifically, these crosslinks are primarily formed between methionine and lysine or hydroxylysine residues and can occur within a single collagen fibril or between different collagen fibrils. Due to its significance as the major crosslink in the collagen IV network, the sulfilimine bond plays critical roles in tissue development and various human diseases. While the proposed reaction mechanism for sulfilimine bond formation is supported by experimental evidence, the precise nature of this bond remained uncertain until computational studies were conducted. The process involves the reaction of hypohalous acids (e.g., HOBr, HOCl), produced by a peroxidasin enzyme in the basement membrane, with the sidechain sulfur of methionine or sidechain nitrogen of lysine/hydroxylysine residues in collagen IV, to form halosulfonium or haloamine intermediates, respectively. The halosulfonium/haloamine then reacts with the sidechain amine/sulfide of the lysine (or hydroxylysine) or methionine respectively, eventually resulting in the formation of the sulfilimine (MetSNLys/Hyl) crosslink. The sulfilimine product formed not only plays a crucial role in physiological processes but also finds applications in various industrial and pharmaceutical contexts. In this review, we provide a comprehensive summary of existing studies, including our own research, aimed at understanding the reaction mechanism, protonation states, characteristic nature, and dynamic behavior of the sulfilimine bond in collagen IV. The goal is to offer readers an overview of this critically important biochemical bond.

Computational Insights into Amide Bond Formation Catalyzed by the Condensation Domain of Nonribosomal Peptide Synthetases.

Nonribosomal peptide synthetases (NRPSs) are important enzymes that synthesize an array of nongenetically encoded peptides. The latter have diverse physicochemical properties and roles. NRPSs are modular enzymes in which, for example, the condensation (C-) domain catalyzes the formation of amide bonds. The NRPS tyrocidine synthetase from Brevibacillus brevis is responsible for synthesizing the cyclic-peptide antibiotic tyrocidine. The first step is formation of an amide bond between a proline and phenylalanine which is catalyzed by a C-domain. In this study, a multiscale computational approach (molecular dynamics and QM/MM) has been used to investigate substrate binding and catalytic mechanism of the C-domain of tyrocidine synthetase. Overall, the mechanism is found to proceed through three exergonic steps in which an active site Histidine, His222, acts as a base and acid. First, His222 acts as a base to facilitate nucleophilic attack of the prolyl nitrogen at the phenylalanyl's carbonyl carbon. This is also the rate-limiting step with a free energy barrier of 38.8 kJ mol-1. The second step is collapse of the resulting tetrahedral intermediate with cleavage of the S-C bond between the phenylalanyl and its Ppant arm, along with formation of the above amide bond. Meanwhile, the now protonated His222 imidazole has rotated toward the newly formed thiolate of the Ppant arm. In the final step, His222 acts as an acid, protonating the thiolate and regenerating a neutral His222. The overall mechanism is found to be exergonic with the final product complex being 46.3 kJ mol-1 lower in energy than the initial reactant complex.

QM/MM investigation of the discriminatory pre-transfer editing mechanism operated by Lysyl-tRNA synthetase.

Aminoacyl-tRNA synthetases (aaRSs) are essential enzymes that remarkable facilitate the aminoacylation process during translation. With a high fidelity, the mischarged tRNA is prevented through implementing pre- and post-transfer proofreading mechanisms. For instance, Lysine-tRNA synthetase charges the native substrate, lysine, to its cognate tRNA. In spite of the great structural similarity between lysine to the noncognate and toxic ornithine, with the side chain of lysine being only one methylene group longer, LysRS is able to achieve this discrimination with a high efficiency. In this work, the hybrid quantum mechanics/molecular mechanics (QM/MM) investigation was applied to probe the pre-transfer editing mechanism catalyzed by lysyl-tRNA synthetase to reject the noncognte aminoacyl, L-ornityl (Orn), compared to the cognate substrate, L-lysyl. Particularly, the self-cyclization pre-transfer editing mechanism was explored for the two substrates. The substrate-assisted self-cyclization editing of Orn-AMP, where its phosphate moiety acts as the catalytic base, is found to be the rate-determining step with an energy barrier of 101.2 kJ mol-1. Meanwhile, the corresponding rate-limiting pathway for the native Lys-AMP lies at 140.2 kJ mol-1. This observation clearly indicated the infeasibility of this catalytic scenario in the presence of the native substrate. Interestingly, a thermodynamically favorable cyclic product of -92.9 kJ mol-1 with respect to the aminoacyl reactant complex demonstrated evidence of a successful pre-transfer editing. This reaction resulted in the discharge of the on-cognate -ornithine derivative from LysU's active site. These valuable mechanistic insights are valuable to enrich our knowledge of this extremely efficient and specific catalytic machinery of LysRS.Communicated by Ramaswamy H. Sarma.

Computational Insights into Protein Aging: Spontaneous Deamidation of Glutamine.

Spontaneous deamidation of amino acids is a physiologically important process, particularly for protein aging and diseases. Despite its widespread occurrence, the mechanism of glutamine deamidation particularly within proteins remains poorly understood. We have used a multiscale computational approach to investigate glutamine deamidation in the tripeptide Glycine-Glutamine-Glycine (Gly-Gln-Gly) and γS-Crystallin protein. Specifically, both the 5- and 6-membered water-assisted deamidation pathways in the tripeptide have been elucidated and compared. Both are found to occur in three stages: iminol formation, cyclization, and deamination. The rate-limiting step in each mechanism is nucleophilic attack of the backbone iminol nitrogen, formed in the first stage, at the glutamine's side-chain carbonyl carbon. For the 6- and 5-membered mechanisms, this occurs with a free energy cost of 136.4 and 179.5 kJ mol-1, respectively. Thus, overall, in the Gly-Gln-Gly tripeptide, the 6-membered pathway is preferred. Furthermore, the free energies for forming cyclic intermediates and products at selected Gln residues (based on experimentally reported % deamidation) in γS-Crystallin have been obtained. It is found that the 5-membered product complex is exergonic at -25.3 kJ mol-1, while the 6-membered product complex is calculated to be endergonic at 90.7 kJ mol-1. Thus, the deamidation pathway in folded and constrained proteins may not exclusively follow the 6-membered route. Molecular dynamics (MD) simulations of γS-Crystallin indicate that deamidation is more likely to occur when two or more water molecules are in the proximity of the glutamine residue. Consequently, significant conformational changes are found to accompany Gln120 deamidation in γS-Crystallin. This in turn can influence water availability at the other Gln residues considered and hence potentially their deamidation. Collectively, these results provide comprehensive insights into spontaneous water-assisted deamidation of glutamine residues in peptides and into the role and impact of Gln deamidation in proteins.

Uncovering Structure-Activity Relationships of Phenethylamines: Paving the Way for Innovative Mental Health Treatments.

The rapidly evolving psychedelic industry has garnered considerable attention due to 3,4-methylenedioxymethamphetamine-assisted psychotherapy's ground-breaking success in treating moderate-to-severe Post-traumatic Stress Disorder in two Phase 3 clinical trials. This has opened Pandora's box for the development of innovative therapeutic modalities. Of particular interest are the phenethylamines and their ability to inhibit monoamine transporters. In this study, we employed the quantitative structure-activity relationship methodology to develop three vigorous models for the reuptake of serotonin, dopamine, and norepinephrine through monoamine transporters. These models were thoroughly validated using various criteria, including fitting (R2DAT = 0.869, R2SERT = 0.828, and R2NET = 0.887), internal (Q2looDAT = 0.795, Q2looSERT = 0.784, and Q2looNET = 0.820), and external (RMSEextDAT = 0.373, R2extDAT = 0.831, RMSEextSERT = 0.200, R2extSERT = 0.955, RMSEextNET = 0.318, and R2extNET = 0.711) criteria.