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 impacts of these mutations are 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 treating cancers, Traboulsi syndrome and, CKD.

Moringa oleifera-based feed supplement protects against renal ischaemia/reperfusion injury via downregulation of Bax/caspase 3 signaling.

Introduction: Ischaemia/reperfusion (I/R) may lead to acute kidney injury via the induction of oxidative stress. On the other hand, Moringa oleifera has been reported to exert antioxidant activities. This study was designed to assess whether or not Moringa oleifera-based feed supplement could prevent I/R-induced renal injury. Materials and methods: Renal I/R was induced by occluding the right renal artery for 30 min followed by a 2-h reperfusion. Results: Renal I/R led to increased absolute renal weight and renal organo-somatic weight index. Renal I/R also caused distortion of renal histoarchitecture and impaired renal function evidenced by elevated serum creatinine and blood urea nitrogen. In addition, renal I/R significantly elevated renal levels of hydrogen peroxide, MDA, and advanced oxidation protein products, but suppressed the levels of reduced glutathione, protein thiol, and non-protein thiol, and the activities of superoxide dismutase and glutathione peroxidase. In addition, renal I/R up-regulated myeloperoxidase activity and the renal levels of NO, TNF-α, and IL-6. Renal I/R also up-regulated Bax and caspase 3 expression in the kidney. Furthermore, I/R-driven structural and biochemical alterations were markedly inhibited by Moringa oleifera-based feed supplement. Discussion: These results suggest that Moringa oleifera-based feed supplement may preserve the gross and histoarchitectural integrity of the kidney as well as renal function via downregulation of Bax/caspase 3 signaling by targeting oxidative stress, inflammation and apoptosis in the kidney of I/R rat.

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.

Catalytic Mechanism of Collagen Hydrolysis by Zinc(II)-Dependent Matrix Metalloproteinase-1.

Human matrix metalloproteinase-1 (MMP-1) is a zinc(II)-dependent enzyme that catalyzes collagenolysis. Despite the availability of extensive experimental data, the mechanism of MMP-1-catalyzed collagenolysis remains poorly understood due to the lack of experimental structure of a catalytically productive enzyme-substrate complex of MMP-1. In this study, we apply molecular dynamics and combined quantum mechanics/molecular mechanics to reveal the reaction mechanism of MMP-1 based on a computationally modeled structure of the catalytically competent complex of MMP-1 that contains a large triple-helical peptide substrate. Our proposed mechanism involves the participation of an auxiliary (second) water molecule (wat2) in addition to the zinc(II)-coordinated water (wat1). The reaction initiates through a proton transfer to Glu219, followed by a nucleophilic attack by a zinc(II)-coordinated hydroxide anion nucleophile at the carbonyl carbon of the scissile bond, leading to the formation of a tetrahedral intermediate (IM2). The process continues with a hydrogen-bond rearrangement to facilitate proton transfer from wat2 to the amide nitrogen of the scissile bond and, finally, C-N bond cleavage. The calculations indicate that the rate-determining step is the water-mediated nucleophilic attack with an activation energy barrier of 22.3 kcal/mol. Furthermore, the calculations show that the hydrogen-bond rearrangement/proton-transfer step can proceed in a consecutive or concerted manner, depending on the conformation of the tetrahedral intermediate, with the consecutive mechanism being energetically preferable. Overall, the study reveals the crucial role of a second water molecule and the dynamics for effective MMP-1-catalyzed collagenolysis.

Comprehensive review of current management guidelines of chronic kidney disease.

Chronic kidney disease (CKD) is a prevalent and progressive condition affecting millions worldwide. It is a long-term condition characterized by gradual loss of kidney function over time. The management of CKD is complex and requires a multidisciplinary approach. This review aims to outline the current management guidelines for CKD. The study included a comprehensive search of various PubMed, Embase, and the Cochrane Library databases for articles published between 2010 and 2023. The search terms used were "chronic kidney disease," "management," and "guidelines." The inclusion criteria were articles that provided management guidelines for patients with CKD. A total of 23 articles were included in the review. Most articles were based on the Kidney Disease Improving Global Outcomes guidelines, the most widely recognized and used guidelines for managing CKD. The study found that the guidelines emphasize the importance of early detection and management of CKD and the need for an approach that involves multiple disciplines in its management. The guidelines recommend several interventions to slow the progression of CKD, including blood pressure control, glycemic control in diabetic patients, and reduce proteinuria. Other interventions include lifestyle modifications such as dietary changes, physical activity, and smoking cessation. The guidelines also recommend regular monitoring of kidney function and referral to a nephrologist for patients with advanced CKD or other complications. Overall, the current management guidelines for CKD emphasize the importance of early detection and a multidisciplinary approach to its management.

Protein-protein interaction and interference of carcinogenesis by supramolecular modifications.

Protein-protein interactions (PPIs) are essential in normal biological processes, but they can become disrupted or imbalanced in cancer. Various technological advancements have led to an increase in the number of PPI inhibitors, which target hubs in cancer cell's protein networks. However, it remains difficult to develop PPI inhibitors with desired potency and specificity. Supramolecular chemistry has only lately become recognized as a promising method to modify protein activities. In this review, we highlight recent advances in the use of supramolecular modification approaches in cancer therapy. We make special note of efforts to apply supramolecular modifications, such as molecular tweezers, to targeting the nuclear export signal (NES), which can be used to attenuate signaling processes in carcinogenesis. Finally, we discuss the strengths and weaknesses of using supramolecular approaches to targeting PPIs.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

The front cover artwork is provided by Dr. Karabencheva-Christova's group at Michigan Technological University. The images show the initially formed and the catalytically productive conformations of MMP-1 complex with the Triple Helical Peptide (THP), the free energy profile connecting them as well as the coordination geometry of the catalytic zinc (II). The background shows the collagen macromolecule. Read the full text of the Research Article at 10.1002/cphc.202200649.

Identification of HER2 inhibitors from curcumin derivatives using combination of in silico screening and molecular dynamics simulation.

Breast cancer remains a major world health challenge in women. Some Breast cancers are human epidermal growth factor receptor 2 (HER2) positive. Since this protein promotes the growth of cancer cells, it remains a therapeutic target for novel drugs. This study uses in silico model to predict HER2 inhibitors from curcumin derivatives via QSAR, e-pharmacophore, ADMET as well as structure-based virtual screening using Schrodinger suite. The molecular dynamics simulation of lead compounds, reference ligand and co-crystalized ligand was performed using GROMACS. At the end, eight active curcumin derivatives were predicted as inhibitors of HER2 with high binding affinity and better interaction compared with the reference drug (Neratinib) but lower binding affinity compared with the co-crystalized ligand (TAK-285). After prediction of the bioactivity of the molecules using AutoQSAR, the hit compounds showed appreciable inhibitory pIC50 compared with the reference and co-crystalized ligands against HER2. The pharmacokinetics profile predicted the eight hit compounds as drug-like and drug candidates. The MD simulation predicted the stability of the two top-scored compounds (10763284 and 78321412) in complex with HER2 for the final 80 ns of the trajectory period after initial equilibration with higher H-bond interactions in the protein-reference drug complex compared to the hit compounds-HER2 complexes. This study revealed that curcumin derivatives especially (1E,6E)-1,8-bis(4-hydroxy-3-methoxyphenyl)octa-1,6-diene-3,5-dione and (1E,6E)-4-ethyl-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-dione were identified to demonstrate inhibitory activity against HER2 which is comparable to neratinib. Conclusively, the lead compounds require further in vitro and in vivo experimental validation for the discovery of new HER2 antagonists for breast cancer management.Communicated by Ramaswamy H. Sarma.

Mechanism of the Early Catalytic Events in the Collagenolysis by Matrix Metalloproteinase-1.

Metalloproteinase-1 (MMP-1) catalyzed collagen degradation is essential for a wide variety of normal physiological processes, while at the same time contributing to several diseases in humans. Therefore, a comprehensive understanding of this process is of great importance. Although crystallographic and spectroscopic studies provided fundamental information about the structure and function of MMP-1, the precise mechanism of collagen degradation especially considering the complex and flexible structure of the substrate, remains poorly understood. In addition, how the protein environment dynamically reorganizes at the atomic scale into a catalytically active state capable of collagen hydrolysis remains unknown. In this study, we applied experimentally-guided multiscale molecular modeling methods including classical molecular dynamics (MD), well-tempered (WT) classical metadynamics (MetD), combined quantum mechanics/molecular mechanics (QM/MM) MD and QM/MM MetD simulations to explore and characterize the early catalytic events of MMP-1 collagenolysis. Importantly the study provided a complete atomic and dynamic description of the transition from the open to the closed form of the MMP-1•THP complex. Notably, the formation of catalytically active Michaelis complex competent for collagen cleavage was characterized. The study identified the changes in the coordination state of the catalytic zinc(II) associated with the conformational transformation and the formation of catalytically productive ES complex. Our results confirm the essential role of the MMP-1 catalytic domain's α-helices (hA, hB and hC) and the linker region in the transition to the catalytically competent ES complex. Overall, the results provide unique mechanistic insight into the conformational transformations and associated changes in the coordination state of the catalytic zinc(II) that would be important for the design of effective MMP-1 inhibitors.

Mechanisms underpinning Carpolobia lutea G. Don ethanol extract's neurorestorative and antipsychotic-like activities in an NMDA receptor antagonist model of schizophrenia.

ETHNOPHARMACOLOGICAL RELEVANCE: Persistent ketamine insults to the central nervous system block NMDA receptors and disrupt putative neurotransmission, oxido-nitrosative, and inflammatory pathways, resulting in schizophrenia-like symptoms in animals. Previously, the ethnomedicinal benefits of Carpolobia lutea against insomnia, migraine headache, and insanity has been documented, but the mechanisms of action remain incomplete. AIM OF THE STUDY: Presently, we explored the neuro-therapeutic role of Carpolobia lutea ethanol extract (C. lutea) in ketamine-induced schizophrenia-like symptoms in mice. MATERIALS AND METHODS: Sixty-four male Swiss (22 ± 2 g) mice were randomly assigned into eight groups (n = 8/group) and exposed to a reversal ketamine model of schizophrenia. For 14 days, either distilled water (10 mL/kg; p.o.) or ketamine (20 mg/kg; i.p.) was administered, following possible reversal treatments with C. lutea (100, 200, 400, and 800 mg/kg; p.o.), haloperidol (1 mg/kg, p.o.), or clozapine (5 mg/kg; p.o.) beginning on days 8-14. During the experiment, a battery of behavioral characterizations defining schizophrenia-like symptoms were obtained using ANY-maze software, followed by neurochemical, oxido-inflammatory and histological assessments in the mice brains. RESULTS: A 7-day reversal treatment with C. lutea reversed predictors of positive, negative and cognitive symptoms of schizophrenia. C. lutea also mitigated ketamine-induced neurochemical derangements as evidenced by modulations of dopamine, glutamate, norepinephrine and serotonin neurotransmission. Also, the increased acetylcholinesterase activity, malondialdehyde nitrite, interleukin-6 and tumor necrosis-factor-α concentrations were reversed by C. lutea accompanied with elevated levels of catalase, superoxide dismutase and reduced glutathione. Furthermore, C. lutea reversed ketamine-induced neuronal alterations in the prefrontal cortex, hippocampus and cerebellum sections of the brain. CONCLUSION: These findings suggest that C. lutea reverses the cardinal symptoms of ketamine-induced schizophrenia in a dose-dependent fashion by modulating the oxido-inflammatory and neurotransmitter-related mechanisms.

How Human TET2 Enzyme Catalyzes the Oxidation of Unnatural Cytosine Modifications in Double-Stranded DNA.

Methylation of cytosine bases is strongly linked to gene expression, imprinting, aging, and carcinogenesis. The Ten-eleven translocation (TET) family of enzymes, which are Fe(II)/2-oxoglutarate (2OG)-dependent enzymes, employ Fe(IV)=O species to dealkylate the lesioned bases to an unmodified cytosine. Recently, it has been shown that the TET2 enzyme can catalyze promiscuously DNA substrates containing unnatural alkylated cytosine. Such unnatural substrates of TET can be used as direct probes for measuring the TET activity or capturing TET from cellular samples. Herein, we studied the catalytic mechanisms during the oxidation of the unnatural C5-position modifications (5-ethylcytosine (5eC), 5-vinylcytosine (5vC) and 5-ethynylcytosine (5eyC)) and the demethylation of N4-methylated lesions (4-methylcytosine (4mC) and 4,4-dimethylcytosine(4dmC)) of the cytosine base by the TET2 enzyme using molecular dynamics (MD) and combined quantum mechanics and molecular mechanics (QM/MM) computational approaches. The results reveal that the chemical nature of the alkylation of the double-stranded (ds) DNA substrates induces distinct changes in the interactions in the binding site, the second coordination sphere, and long-range correlated motions of the ES complexes. The rate-determining hydrogen atom transfer (HAT) is faster in N4-methyl substituent substrates than in the C5-alkylations. Importantly, the calculations show the preference of hydroxylation over desaturation in both 5eC and 5vC substrates. The studies elucidate the post-hydroxylation rearrangements of the hydroxylated intermediates of 5eyC and 5vC to ketene and 5-formylmethylcytosine (5fmC), respectively, and hydrolysis of hemiaminal intermediate of 4mC to formaldehyde and unmodified cytosine proceed exclusively in aqueous solution outside of the enzyme environment. Overall, the studies show that the chemical nature of the unnatural alkylated cytosine substrates exercises distinct effects on the binding interactions, reaction mechanism, and dynamics of TET2.

Can Second Coordination Sphere and Long-Range Interactions Modulate Hydrogen Atom Transfer in a Non-Heme Fe(II)-Dependent Histone Demethylase?

Fe(II)-dependent oxygenases employ hydrogen atom transfer (HAT) to produce a myriad of products. Understanding how such enzymes use dynamic processes beyond the immediate vicinity of the active site to control the selectivity and efficiency of HAT is important for metalloenzyme engineering; however, obtaining such knowledge by experiments is challenging. This study develops a computational framework for identifying second coordination sphere (SCS) and especially long-range (LR) residues relevant for catalysis through dynamic cross-correlation analysis (DCCA) using the human histone demethylase PHF8 (KDM7B) as a model oxygenase. Furthermore, the study explores the mechanistic pathways of influence of the SCS and LR residues on the HAT reaction. To demonstrate the plausibility of the approach, we investigated the effect of a PHF8 F279S clinical mutation associated with X-linked intellectual disability, which has been experimentally shown to ablate PHF8-catalyzed demethylation. In agreement, the molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) studies showed a change in the H31-14K9me2 substrate orientation and an increased HAT barrier. We systematically analyzed the pathways by which the identified SCS and LR residues may influence HAT by exploring changes in H3K9me2 substrate orientation, interdomain correlated motions, HAT transition state stabilization, reaction energetics, electron transfer mechanism, and alterations in the intrinsic electric field of PHF8. Importantly, SCS and LR variations decrease key motions of α9-α12 of the JmjC domain toward the Fe(IV)-center that are associated with tighter binding of the H31-14K9me2 substrate. SCS and LR residues alter the intrinsic electric field of the enzyme along the reaction coordinate and change the individual energetic contributions of residues toward TS stabilization. The overall results suggest that DCCA can indeed identify non-active-site residues relevant for catalysis. The substitutions of such dynamically correlated residues might be used as a tool to tune HAT in non-heme Fe(II)- and 2OG-dependent enzymes.

YfeX - A New Platform for Carbene Transferase Development with High Intrinsic Reactivity.

Carbene transfer biocatalysis has evolved from basic science to an area with vast potential for the development of new industrial processes. In this study, we show that YfeX, naturally a peroxidase, has great potential for the development of new carbene transferases, due to its high intrinsic reactivity, especially for the N-H insertion reaction of aromatic and aliphatic primary and secondary amines. YfeX shows high stability against organic solvents (methanol and DMSO), greatly improving turnover of hydrophobic substrates. Interestingly, in styrene cyclopropanation, WT YfeX naturally shows high enantioselectivity, generating the trans product with 87 % selectivity for the (R,R) enantiomer. WT YfeX also catalyzes the Si-H insertion efficiently. Steric effects in the active site were further explored using the R232A variant. Quantum Mechanics/Molecular Mechanics (QM/MM) calculations reveal details on the mechanism of Si-H insertion. YfeX, and potentially other peroxidases, are exciting new targets for the development of improved carbene transferases.

Will Africans take COVID-19 vaccination?

The economic and humanistic impact of COVID-19 pandemic is enormous globally. No definitive treatment exists, hence accelerated development and approval of COVID-19 vaccines, offers a unique opportunity for COVID-19 prevention and control. Vaccine hesitancy may limit the success of vaccine distribution in Africa, therefore we assessed the potentials for coronavirus vaccine hesitancy and its determinants among Africans. An online cross-sectional African-wide survey was administered in Arabic, English, and French languages. Questions on demographics, self-reported health status, vaccine literacy, knowledge and perception on vaccines, past experience, behavior, infection risk, willingness to receive and affordability of the SARS-COV-2 vaccine were asked. Data were subjected to descriptive and inferential statistics. A total of 5,416 individuals completed the survey. Approximately, 94% were residents of 34 African countries while the other Africans live in the Diaspora. Only 63% of all participants surveyed were willing to receive the COVID-19 vaccination as soon as possible and 79% were worried about its side effects. Thirty-nine percent expressed concerns of vaccine-associated infection. The odds of vaccine hesitancy was 0.28 (95% CI: 0.22, 0.30) among those who believed their risk of infection was very high, compared to those who believed otherwise. The odds of vaccine hesitancy was one-fifth (OR = 0.21, 95% CI: 0.16, 0.28) among those who believed their risk of falling sick was very high, compared to those who believed their risk of falling very sick was very low. The OR of vaccine hesitancy was 2.72 (95% CI: 2.24, 3.31) among those who have previously refused a vaccine for themselves or their child compared to counterparts with no self-reported history of vaccine hesitancy. Participants want the vaccines to be mandatory (40%), provided free of charge (78%) and distributed in homes and offices (44%). COVID-19 vaccine hesitancy is substantial among Africans based on perceived risk of coronavirus infection and past experiences.

What Is the Catalytic Mechanism of Enzymatic Histone N-Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field?

Invited for the cover of this issue are Christo Z. Christov and co-workers at Michigan Technological University and University of Oxford. The image depicts the effects of applying an external electric field on the demethylation of dimethylated arginine substrate by a non-heme Fe center Histone N-methyl arginine demethylase. Read the full text of the article at 10.1002/chem.202101174.

What Is the Catalytic Mechanism of Enzymatic Histone N-Methyl Arginine Demethylation and Can It Be Influenced by an External Electric Field?

Arginine methylation is an important mechanism of epigenetic regulation. Some Fe(II) and 2-oxoglutarate dependent Jumonji-C (JmjC) Nϵ-methyl lysine histone demethylases also have N-methyl arginine demethylase activity. We report combined molecular dynamic (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) studies on the mechanism of N-methyl arginine demethylation by human KDM4E and compare the results with those reported for N-methyl lysine demethylation by KDM4A. At the KDM4E active site, Glu191, Asn291, and Ser197 form a conserved scaffold that restricts substrate dynamics; substrate binding is also mediated by an out of active site hydrogen-bond between the substrate Ser1 and Tyr178. The calculations imply that in either C-H or N-H potential bond cleaving pathways for hydrogen atom transfer (HAT) during N-methyl arginine demethylation, electron transfer occurs via a σ-channel; the transition state for the N-H pathway is ∼10 kcal/mol higher than for the C-H pathway due to the higher bond dissociation energy of the N-H bond. The results of applying external electric fields (EEFs) reveal EEFs with positive field strengths parallel to the Fe=O bond have a significant barrier-lowering effect on the C-H pathway, by contrast, such EEFs inhibit the N-H activation rate. The overall results imply that KDM4 catalyzed N-methyl arginine demethylation and N-methyl lysine demethylation occur via similar C-H abstraction and rebound mechanisms leading to methyl group hydroxylation, though there are differences in the interactions leading to productive binding of intermediates.

Moringa oleifera Extract Extenuates Echis ocellatus Venom-Induced Toxicities, Histopathological Impairments and Inflammation via Enhancement of Nrf2 Expression in Rats.

Echis ocellatus snakebite causes more fatalities than all other African snake species combined. Moringa oleifera reportedly possesses an antivenom property. Therefore, we evaluated the effectiveness of M. oleifera ethanol extract (MOE) against E. ocellatus venom (EOV) toxicities. Thirty male rats were grouped as follows (n = 5): Group 1 (normal control received saline), groups 2 to 6 were administered intraperitoneally, 0.22 mg/kg (LD50) of EOV. Group 2 was left untreated while group 3 to 6 were treated post-envenoming with 0.2 mL of polyvalent antivenom, 200, 400, and 600 mg/kg of MOE respectively. MOE significantly (p < 0.05) normalized the altered haematological indices and blood electrolytes profiles. MOE attenuated venom-induced cellular dysfunctions, characterized by a significant increase in NRF2, and concomitant downregulation of increased antioxidant enzymes (SOD and CAT) activities in the serum and heart of the treated rats. MOE normalized the elevated TNF-α and IL-1ß in serum and heart tissues. Furthermore, the IgG titre value was significantly (p < 0.5) higher in the envenomed untreated group compared to the MOE-treated groups. Hemorrhagic, hemolytic and coagulant activities of the venom were strongly inhibited by the MOE dose, dependently. Lesions noticed on tissues of vital organs of untreated rats were abolished by MOE. Our findings substantiate the effectiveness of MOE as a potential remedy against EOV toxicities.

Role of Structural Dynamics in Selectivity and Mechanism of Non-heme Fe(II) and 2-Oxoglutarate-Dependent Oxygenases Involved in DNA Repair.

AlkB and its human homologue AlkBH2 are Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenases that repair alkylated DNA bases occurring as a consequence of reactions with mutagenic agents. We used molecular dynamics (MD) and combined quantum mechanics/molecular mechanics (QM/MM) methods to investigate how structural dynamics influences the selectivity and mechanisms of the AlkB- and AlkBH2-catalyzed demethylation of 3-methylcytosine (m3C) in single (ssDNA) and double (dsDNA) stranded DNA. Dynamics studies reveal the importance of the flexibility in both the protein and DNA components in determining the preferences of AlkB for ssDNA and of AlkBH2 for dsDNA. Correlated motions, including of a hydrophobic ß-hairpin, are involved in substrate binding in AlkBH2-dsDNA. The calculations reveal that 2OG rearrangement prior to binding of dioxygen to the active site Fe is preferred over a ferryl rearrangement to form a catalytically productive Fe(IV)=O intermediate. Hydrogen atom transfer proceeds via a σ-channel in AlkBH2-dsDNA and AlkB-dsDNA; in AlkB-ssDNA, there is a competition between σ- and π-channels, implying that the nature of the complexed DNA has potential to alter molecular orbital interactions during the substrate oxidation. Our results reveal the importance of the overall protein-DNA complex in determining selectivity and how the nature of the substrate impacts the mechanism.

Structure-function relationships in KDM7 histone demethylases.

The demethylation of lysine residues of histone proteins is a key epigenetic mechanism in cells. The enzymes that catalyze these processes are called histone demethylases (KDMs). The largest family of KDMs is the Jumonji C (JmjC) domain-containing enzymes; these includes KDM2-7 subfamily of enzymes. The JmjC proteins are Fe(II) and 2-Oxoglutarate (2OG) - dependent dioxygenases that couple substrate oxidation to decarboxylation of 2OG to form succinate and CO2. The KDM7 subfamily of enzymes - PHF8 (KDM7B) and KIAA1718 (KDM7A) are human JmjC 2OG-dependent Nε-methyl lysine demethylases and are involved in demethylation of lysine residues in histones such as H3K27me2/1, H3K9me2/1 and H4K20me1. These enzymes are involved in multiple pathologic processes, including cancers and mental retardation. In this chapter, we present the current state of the art in the structural, biochemical and computational studies of KDM7 enzymes.