Novel Insights into the Catalytic Mechanism of Collagenolysis by Zn(II)-Dependent Matrix Metalloproteinase-1.

Collagen hydrolysis, catalyzed by Zn(II)-dependent matrix metalloproteinases (MMPs), is a critical physiological process. Despite previous computational investigations into the catalytic mechanisms of MMP-mediated collagenolysis, a significant knowledge gap in understanding remains regarding the influence of conformational sampling and entropic contributions at physiological temperature on enzymatic collagenolysis. In our comprehensive multilevel computational study, employing quantum mechanics/molecular mechanics (QM/MM) metadynamics (MetD) simulations, we aimed to bridge this gap and provide valuable insights into the catalytic mechanism of MMP-1. Specifically, we compared the full enzyme-substrate complex in solution, clusters in solution, and gas-phase to elucidate insights into MMP-1-catalyzed collagenolysis. Our findings reveal significant differences in the catalytic mechanism when considering thermal effects and the dynamic evolution of the system, contrasting with conventional static potential energy surface QM/MM reaction path studies. Notably, we observed a significant stabilization of the critical tetrahedral intermediate, attributed to contributions from conformational flexibility and entropy. Moreover, we found that protonation of the scissile bond nitrogen occurs via proton transfer from a Zn(II)-coordinated hydroxide rather than from a solvent water molecule. Following C-N bond cleavage, the C-terminus remains coordinated to the catalytic Zn(II), while the N-terminus forms a hydrogen bond with a solvent water molecule. Subsequently, the release of the C-terminus is facilitated by the coordination of a water molecule. Our study underscores the pivotal role of protein conformational dynamics at physiological temperature in stabilizing the transition state of the rate-limiting step and key intermediates, compared to the corresponding reaction in solution. These fundamental insights into the mechanism of collagen degradation provide valuable guidance for the development of MMP-1-specific inhibitors.

Effects of Clinical Mutations in the Second Coordination Sphere and Remote Regions on the Catalytic Mechanism of Non-Heme Fe(II)/2-Oxoglutarate-Dependent Aspartyl Hydroxylase AspH.

Aspartyl/asparaginyl hydroxylase (AspH) catalyzes the post-translational hydroxylations of vital human proteins, playing an essential role in maintaining their biological functions. Single-point mutations in the Second Coordination Sphere (SCS) and long-range (LR) residues of AspH have been linked to pathological conditions such as the ophthalmologic condition Traboulsi syndrome and chronic kidney disease (CKD). Although the clinical impact of these mutations is established, there is a critical knowledge gap regarding their specific atomistic effects on the catalytic mechanism of AspH. In this study, we report integrated computational investigations on the potential mechanistic implications of four mutant forms of human AspH with clinical importance: R735W, R735Q, R688Q, and G434V. All the mutant forms exhibited altered binding interactions with the co-substrate 2-oxoglutarate (2OG) and the main substrate in the ferric-superoxo and ferryl complexes, which are critical for catalysis, compared to the wild-type (WT). Importantly, the mutations strongly influence the energetics of the frontier molecular orbitals (FMOs) and, thereby, the activation energies for the hydrogen atom transfer (HAT) step compared to the WT AspH. Insights from our study can contribute to enzyme engineering and the development of selective modulators for WT and mutants of AspH, ultimately aiding in the treatment of Traboulsi syndrome and CKD.

Unusual catalytic strategy by non-heme Fe(ii)/2-oxoglutarate-dependent aspartyl hydroxylase AspH.

Biocatalytic C-H oxidation reactions are of important synthetic utility, provide a sustainable route for selective synthesis of important organic molecules, and are an integral part of fundamental cell processes. The multidomain non-heme Fe(ii)/2-oxoglutarate (2OG) dependent oxygenase AspH catalyzes stereoselective (3R)-hydroxylation of aspartyl- and asparaginyl-residues. Unusually, compared to other 2OG hydroxylases, crystallography has shown that AspH lacks the carboxylate residue of the characteristic two-His-one-Asp/Glu Fe-binding triad. Instead, AspH has a water molecule that coordinates Fe(ii) in the coordination position usually occupied by the Asp/Glu carboxylate. Molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies reveal that the iron coordinating water is stabilized by hydrogen bonding with a second coordination sphere (SCS) carboxylate residue Asp721, an arrangement that helps maintain the six coordinated Fe(ii) distorted octahedral coordination geometry and enable catalysis. AspH catalysis follows a dioxygen activation-hydrogen atom transfer (HAT)-rebound hydroxylation mechanism, unusually exhibiting higher activation energy for rebound hydroxylation than for HAT, indicating that the rebound step may be rate-limiting. The HAT step, along with substrate positioning modulated by the non-covalent interactions with SCS residues (Arg688, Arg686, Lys666, Asp721, and Gln664), are essential in determining stereoselectivity, which likely proceeds with retention of configuration. The tetratricopeptide repeat (TPR) domain of AspH influences substrate binding and manifests dynamic motions during catalysis, an observation of interest with respect to other 2OG oxygenases with TPR domains. The results provide unique insights into how non-heme Fe(ii) oxygenases can effectively catalyze stereoselective hydroxylation using only two enzyme-derived Fe-ligating residues, potentially guiding enzyme engineering for stereoselective biocatalysis, thus advancing the development of non-heme Fe(ii) based biomimetic C-H oxidation catalysts, and supporting the proposal that the 2OG oxygenase superfamily may be larger than once perceived.

Comprehensive computational study on reaction mechanism of N-Nitroso dimethyl amine formation from substituted hydrazine derivatives during ozonation.

N- Nitrosodimethyl amine, the simplest member of the N-Nitrosamine family, is a carcinogenic and mutagenic agent that has gained considerable research interest owing to its toxic nature. Ozonation of industrially important hydrazines, such as unsymmetrical dimethylhydrazine (UDMH) or monomethylhydrazine (MMH), has been associated with NDMA formation and accumulation in the environment. UDMH/MMH - ozonation also leads to several other transformation products such as acetaldehyde dimethyl hydrazine (ADMH), tetramethyl tetra azene (TMT), diazomethane, methyl diazene, etc, which can be either precursors or competitors for NDMA formation. However, the relevant chemistry detailing the formation of these transformation products from UDMH/MMH -ozone reaction and their subsequent conversion to NDMA is not well understood. In this work, we explored the formation mechanism of ADMH and TMT from UDMH-ozonation and their further oxidation to NDMA using the second-order Moller Plesset perturbation theory employing the 6-311G(d) basis set. We have also investigated how MMH selectively forms methyl diazene and diazomethane under normal conditions and NDMA in the presence of excess ozone. Our calculations indicate that the reactions proceed via an initial H abstraction from the hydrazine -NH2 group, followed by the oxidation of the generated N-radical species. The formation of ADMH from the UDMH-ozone reaction involves an acetaldehyde intermediate, which then reacts with a second UDMH molecule to generate ADMH. The preferable attack of ozone molecule on N=C bond of ADMH generates DMAN intermediate, which subsequently undergoes oxidation to form NDMA. Unlike other transformation products, TMT formation occurs via the dimerization of DMAN. 1Though there exists an N=N bond in the TMT, which are preferable attacking sites for ozone, experimental studies show the lower yields of NDMA formation, which corroborates with the high activation barrier required for the process (42 kcal/mol). Overall, our calculated results agree well with the experimental observations and rate constants. Computational calculations bring new insights into the electronic nature and kinetics of the elementary reactions of this pathway, enabled by computed energies of structures that are not possible to access experimentally.

A Quantum Chemical Investigation into the Molecular Mechanism of the Atmospheric Reactions of Chemi-Ions with Nitrogen and Nitrogen Oxides.

Nitrogen oxides and chemi-ions are atmospheric pollutants with considerable aeronomic interest. These toxicants can react with each other, producing various ionic species and highly reactive by-products that play a crucial role in aerosol clustering and mediate several important atmospheric reactions. Understanding the chemical reactivity of these pollutants can provide essential information for controlling their excess emission into the atmosphere. Computational modeling and electronic structure studies help in predicting the structure, reactivity, and thermodynamics of transient atmospheric chemical species and can guide experimental research by providing vital mechanistic insights and data. In the present study, a computational investigation into the mechanisms of the binary associative reactions between negative ions: O2- and O3- with NO, NO2, and N2 was conducted using the Coupled-Cluster Singles and Doubles (CCSD) theory. Five model reactions between N2/NOx with On- (n = 2, 3) were considered in this work. Our calculations revealed that reactions (2) and (5) are two sequential processes involving intermediates, and all others occur in a concerted manner by direct transitions from the reactants to the products, with no isolable intermediates proceeding via single non-planar transition states. Our study revealed that the higher activation barrier required for the formation of NO3- (2) as compared to NO2- (1) could be the reason for the excess formation of NO2- ions over NO3- ions in the atmosphere. Further, all the investigated reactions except (5) are found to be feasible at room temperature. The energy required to break N-N bonds in the N2 molecule justifies the high barrier for (5). The results obtained from the study are in close agreement with the available experimental data. Moreover, the data from the study can be utilized for the evaluation of experiments and model predictions pertaining to NOx oxidation and molecular modeling of the gas-phase chemistry of pollutants/nucleation precursors formed in the Earth's atmosphere and aircraft engines.

A detailed theoretical investigation to unravel the molecular mechanism of the ligand-free copper-catalyzed Suzuki cross-coupling reaction.

The Suzuki-Miyaura coupling (SMC) represents a very efficacious method for constructing C-C bonds in organic synthesis. The ligand-free variants of SMC have been grabbing attention these days. Despite this momentousness, the mechanistic details of the ligand-free variants are scant in the literature. Herein, we have carried out a detailed mechanistic investigation into the ligand-free Cu-catalyzed SMC of unsaturated organic halides with aryl boronic acid with the aid of density functional theory (DFT) calculations employing the conductor-like polarizable continuum model (CPCM) method. The present study elucidates that in the absence of ancillary ligands on the metal, the substrates, base, and solvent molecules could act as pseudo-ancillary ligands to facilitate the cross-coupling reaction. The investigation further revealed that unsaturated halides like alkynyl halides/vinyl halides could act as good ancillary ligands for copper by forming a Cu-π intermediate and promoting a facile transmetalation process. However, regarding the oxidative addition and reductive elimination steps, a concerted pathway is observed contrary to Pd catalyzed Suzuki coupling, owing to the instability of Cu(III) species and the favourability of Csp2-Csp bond formation. In the whole set of mechanisms explored, oxidative addition/oxidative nucleophilic substitution was the rate-determining step in all the cases. A thermodynamically stable π-coordinated intermediate species where the substrate and base molecule are coordinated to the metal center is identified as the rate-determining species for the ligand-free Suzuki cross-coupling reaction. The presence of the aforesaid intermediate increases the energy span and consequently the activation barrier for the rate-determining step. This study unveiled a theoretical rationale for the high-temperature requirement in the ligand-free Cu-catalyzed SMC reaction.

Development of strontium-doped nano hydroxyapatite dentifrice and compare its remineralising potential with a topical cream containing casein phosphopeptide- amorphous calcium phosphate - An In Vitro study.

AIM: To develop and evaluate the efficacy of synthesised strontium-doped nano hydroxyapatite dentifrice and compare its remineralizing potential with a topical cream containing Casein Phospho Peptide - Amorphous Calcium Phosphate, in remineralizing artificial carious lesion on enamel. MATERIALS AND METHODS: Enamel specimens of 4 x 4 x 1 mm were prepared from 90 freshly extracted teeth. Specimens were divided into 3 groups of 30 samples each, based on the type of dentifrice applied that is a control group (Group I) and two experimental groups (Groups II, III). Surface topography and the calcium/phosphorous ratio of all sound specimen were evaluated using Scanning electron microscope and Energy Dispersive X-ray Analysis (SEM-EDAX). The samples in group I and each of the experimental groups were subjected to demineralisation and the calcium/phosphorous ratio of the demineralized specimen were analysed. The samples were then subjected to remineralisation using different agents in each group. Samples in the control group (Group I) were brushed with a conventional dentifrice. In the experimental groups, Group II topical cream with Casein Phosphopeptide and Amorphous Calcium phosphate (CPP-ACP) was used and in Group III laboratory synthesized Strontium-doped nanohydroxyapatite paste (Sr-nHAP), respectively for 28 consecutive days. The samples in the both the control and the two experimental groups were again subjected to SEM-EDAX analysis to analyse the calcium phosphorus ratio following remineralisation cycle. Groupwise comparison of the data was done with one way ANOVA followed by Tukeys Post hoc Test. RESULTS: Both experimental groups (II, III) showed statistically significant remineralisation potential after demineralisation, compared to the control group I. Intergroup comparison showed that the samples in Group III showed the higher remineralisation potential than Group II and was statistically significant. CONCLUSION: Both CPP- ACP containing tooth cream as well as Sr doped nHAp showed remineralisation potential. Sr doped nanohydroxyapatite showed better remineralisation than CPP ACP and can be considered for enamel repair in incipient carious lesions.

Antimicrobial Effect of Ginger, Garlic, Honey, and Lemon Extracts on Streptococcus mutans.

AIM: The aim of this study was to evaluate the effect of lemon, ginger, garlic, and honey extracts on Streptococcus mutans. MATERIALS AND METHODS: Commercially obtained honey, ginger, garlic, and lemon were included in the study to evaluate its efficacy in isolation and in combination against S. mutans. The efficacies of extracts were tested using well diffusion method, and its effect was evaluated by measuring the zone of inhibition around the well. Antimicrobial activity of the extracts was carried out individually and compared considering triplicates of all the extracts. RESULTS: When individual comparison of the extracts was made, garlic showed greatest antimicrobial activity with a mean zone of inhibition (34.9 ± 0.58 mm) and honey showed least antimicrobial activity (0.5 ± 0.6 mm). When combinations of extracts were tested against S. mutans, lemon and garlic combination showed the greatest zone of inhibition (27.6 ± 0.43 mm) compared with other combinations, and ginger + lemon combinations showed the least zone of inhibition (12.6 ± 0.43 mm). CONCLUSION: This study concluded that garlic showed a greatest antimicrobial effect against S. mutans when compared with other preparations individually and garlic and lemon showed greatest zone of inhibition in combination than other preparations. CLINICAL SIGNIFICANCE: Antibiotics and other chemical agents are mainly used to treat the common dental infections. However, due to the excessive use, it can result in antibiotic resistance. Hence, herbal medicines with medicinal values should be replaced with conventional methods.