Coenzyme Binding Research Articles

Structure, dimeric conformation, and coenzyme versatility of p-hydroxybenzoate hydroxylase from Arthrobacter sp. PAMC25564

p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA). PHBHs are commonly known as homodimers, and the prediction of pyridine nucleotide binding and specificity remains an ongoing focus in this field. Therefore, our study aimed to determine the dimerization interface in AspPHBH from Arthrobacter sp. PAMC25564 and identify the canonical pyridine nucleotide-binding residues, along with coenzyme specificity, through site-directed mutagenesis. The results confirm a functional dimeric assembly from a tetramer that appeared in the crystallographic asymmetric unit identical to that established in previous studies. Furthermore, AspPHBH exhibits coenzyme versatility, utilizing both NADH and NADPH, with a preference for NADH. Rational engineering experiments demonstrated that targeted mutations in coenzyme surrounding residues profoundly impact NADPH binding, leading to nearly abrogated enzymatic activity compared to that of NADH. R50, R273, and S166 emerged as significant residues for NAD(P)H binding, having a near-fatal impact on NADPH binding compared to NADH. Likewise, the E44 residue plays a critical role in determining coenzyme specificity. Overall, our findings contribute to the fundamental understanding of the determinants of PHBH's active dimeric conformation, coenzyme binding and specificity holding promise for biotechnological advancements.

Exploring the therapeutic potential of rutin through investigating its inhibitory mechanism on lactate dehydrogenase: Multi-spectral methods and computer simulation

Lactate dehydrogenase (LDH), a crucial enzyme in anaerobic glycolysis, plays a pivotal role in the energy metabolism of tumor cells, positioning it as a promising target for tumor treatment. Rutin, a plant-based flavonoid, offers benefits like antioxidant, antiapoptotic, and antineoplastic effects. This study employed diverse experiments to investigate the inhibitory mechanism of rutin on LDH through a binding perspective. The outcomes revealed that rutin underwent spontaneous binding within the coenzyme binding site of LDH, leading to the formation of a stable binary complex driven by hydrophobic forces, with hydrogen bonds also contributing significantly to sustaining the stability of the LDH-rutin complex. The binding constant (Ka) for the LDH-rutin system was 2.692 ± 0.015 × 104 M−1 at 298 K. Furthermore, rutin induced the alterations in the secondary structure conformation of LDH, characterized by a decrease in α-helix and an increase in antiparallel and parallel β-sheet, and β-turn. Rutin augmented the stability of coenzyme binding to LDH, which could potentially hinder the conversion process among coenzymes. Specifically, Arg98 in the active site loop of LDH provided essential binding energy contribution in the binding process. These outcomes might explain the dose-dependent inhibition of the catalytic activity of LDH by rutin. Interestingly, both the food additives ascorbic acid and tetrahydrocurcumin could reduce the binding stability of LDH and rutin. Meanwhile, these food additives did not produce positive synergism or antagonism on the rutin binding to LDH. Overall, this research could offer a unique insight into the therapeutic potential and medicinal worth of rutin.

Anticancer activity features of imidazole-based ionic liquids and lysosomotropic detergents: in silico and in vitro studies.

Long-chain imidazole-based ionic liquids (compounds 2, 4, 9) and lysosomotropic detergents (compounds 7, 3, 8) with potent anticancer activity were synthesized. Their inhibitory activities against neuroblastoma and leukaemia cell lines were predicted by the new in silico QSAR models. The cytotoxic activities of the synthesized imidazole derivatives were investigated on the SK-N-DZ (human neuroblastoma) and K-562 (human chronic myeloid leukaemia) cell lines. Compounds 2 and 7 showed the highest in vitro cytotoxic effect on both cancer cell lines. The docking procedure of compounds 2 and 7 into the NAD+ coenzyme binding site of deacetylase Sirtuin-1 (SIRT-1) showed the formation of protein-ligand complexes with calculated binding energies of -8.0 and - 8.1kcal/mol, respectively. The interaction of SIRT1 with compounds 2, 7 and 9 and the interaction of Bromodomain-containing protein 4 (BRD4) with compounds 7 and 9 were also demonstrated by thermal shift assay. Compounds 2, 4, 7 and 9 inhibited SIRT1 deacetylase activity in the SIRT-Glo assay. Compounds 7 and 9 showed a moderate inhibitory activity against Aurora kinase A. In addition, compounds 3, 4, 8 and 9 inhibited the Janus kinase 2 activity. The results obtained showed that long-chain imidazole derivatives exhibited cytotoxic activities on K562 leukaemia and SK-N-DZ neuroblastoma cell lines. Furthermore, these compounds inhibited a panel of molecular targets involved in leukaemia and neuroblastoma tumorigenesis. All these results suggest that both long-chain imidazole-based ionic liquids and lysosomotropic detergents may be an effective alternative for the treatment of neuroblastoma and chronic myeloid leukemia and merit further investigation.

Protein-protein interfaces as druggable targets: a common motif of the pyridoxal-5′-phosphate-dependent enzymes to receive the coenzyme from its producer

Pyridoxal-5′-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP may modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in a multitude of the PLP-dependent enzymes for binding the limited number of the PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5′-phosphate oxidase (PNPO) and the PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the PLP acceptors of folds I and II, while PLPBP - with those of folds III and V. Aligning the sequences and 3D structures of the identified interactors of PdxK and PNPO, we have found a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand and residual loops. Pathogenicity of mutations in human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of the coenzyme binding. The enzyme-specific amino acid residues of the common motif may be used to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

Sequences, phylogeny and evolution of mitochondrial delta-1-pyrroline-5-carboxylate dehydrogenases (ALDH4A1). Evidence for a second locus (ALDH4A2) in Drosophila

ALDH4A1 genes encode mitochondrial enzymes of delta-1-pyrroline-5-carboxylate metabolism, generating glutamate from either proline or ornithine. Analyses were undertaken of several vertebrate and invertebrate genomes using reported human and mouse ALDH4A1 amino acid sequences. ALDH4A1 sequences and structures were highly conserved, including residues involved in catalysis, coenzyme binding and enzyme structure, previously reported for mouse and human ALDH4A1. The human ALDH4A1 gene contained 15 coding exons and was more highly expressed in human liver and kidney cortex. Vertebrate ALDH4A1 mitochondrial leader sequences exhibited diverse sequences. Phylogeny studies supported the appearance of the ALDH4A1 gene in invertebrate evolution which has been conserved and retained throughout subsequent vertebrate evolution as a single ALDH4A1 gene. Exceptions included polyploidy observed for the Atlantic salmon (Salmo salar) and African toad (Xenopus laevis) genes. An examination of ALDH4A1 sequences from related Drosophila species supported the appearance of a second ALDH4A gene (ALDH4A2) and time dependent evolutionary changes over the past 50 million years for both genes.

Identifying structural and dynamic changes during the Biliverdin Reductase B catalytic cycle.

Biliverdin Reductase B (BLVRB) is an NADPH-dependent reductase that catalyzes the reduction of multiple substrates and is therefore considered a critical cellular redox regulator. In this study, we sought to address whether both structural and dynamics changes occur between different intermediates of the catalytic cycle and whether these were relegated to just the active site or the entirety of the enzyme. Through X-ray crystallography, we determined the apo BLVRB structure for the first time, revealing subtle global changes compared to the holo structure and identifying the loss of a critical hydrogen bond that "clamps" the R78-loop over the coenzyme. Amide and Cα chemical shift perturbations were used to identify environmental and secondary structural changes between intermediates, with more distant global changes observed upon coenzyme binding compared to substrate interactions. NMR relaxation rate measurements provided insights into the dynamic behavior of BLVRB during the catalytic cycle. Specifically, the inherently dynamic R78-loop that becomes ordered upon coenzyme binding persists through the catalytic cycle while similar regions experience dynamic exchange. However, the dynamic exchange processes were found to differ through the catalytic cycle with several groups of residues exhibiting similar dynamic responses. Finally, both local and distal structural and dynamic changes occur within BLVRB that are dependent solely on the oxidative state of the coenzyme. Thus, through a comprehensive analysis here, this study revealed structural and dynamic alterations in BLVRB through its catalytic cycle that are not simply relegated to the active site, but instead, are allosterically coupled throughout the enzyme.

Enzymatic Characterization of the Isocitrate Dehydrogenase with Dual Coenzyme Specificity from the Marine Bacterium Umbonibacter marinipuiceus.

Isocitrate dehydrogenase (IDH) can be divided into NAD+-dependent and NADP+-dependent types based on the coenzyme specificity. It is worth noting that some IDHs exhibit dual coenzyme specificity characteristics. Herein, a dual coenzyme-dependent IDH from Umbonibacter Marinipuiceus (UmIDH) was expressed, purified, and identified in detail for the first time. SDS-PAGE and Gel filtration chromatography analyses showed that UmIDH is an 84.7 kDa homodimer in solution. The Km values for NAD+ and NADP+ are 1800.0 ± 64.4 μM and 1167.7 ± 113.0 μM in the presence of Mn2+, respectively. Meanwhile, the catalytic efficiency (kcat/Km) of UmIDH is only 2.3-fold greater for NADP+ than NAD+. The maximal activity for UmIDH occurred at pH 8.5 (with Mn2+) or pH 8.7 (with Mg2+) and at 35 °C (with Mn2+ or Mg2+). Heat inactivation assay revealed that UmIDH sustained 50% of maximal activity after incubation at 57 °C for 20 min with either Mn2+ or Mg2+. Moreover, three putative core coenzyme binding residues (R345, L346, and V352) of UmIDH were evaluated by site-directed mutagenesis. This recent work identified a unique dual coenzyme-dependent IDH and achieved the groundbreaking bidirectional modification of this specific IDH's coenzyme dependence for the first time. This provides not only a reference for the study of dual coenzyme-dependent IDH, but also a basis for the investigation of the coenzyme-specific evolutionary mechanisms of IDH.

Structural and functional studies of Arabidopsis thaliana glutamate dehydrogenase isoform 2 demonstrate enzyme dynamics and identify its calcium binding site

Glutamate dehydrogenase (GDH) is an enzyme at the crossroad of plant nitrogen and carbon metabolism. GDH catalyzes the conversion of 2-oxoglutarate into glutamate (2OG → Glu), utilizing ammonia as cosubstrate and NADH as coenzyme. The GDH reaction is reversible, meaning that the NAD+-dependent reaction (Glu → 2OG) releases ammonia. In Arabidopsis thaliana, three GDH isoforms exist, AtGDH1, AtGDH2, and AtGDH3. The subject of this work is AtGDH2. Previous reports have suggested that enzymes homologous to AtGDH2 contain a calcium-binding EF-hand motif located in the coenzyme binding domain. Here, we show that while AtGDH2 indeed does bind calcium, the binding occurs elsewhere and the region predicted to be the EF-hand motif has a completely different structure. As the true calcium binding site is > 20 Å away from the active site, it seems to play a structural, rather than catalytic role. We also performed comparative kinetic characterization of AtGDH1 and AtGDH2 using spectroscopic methods and isothermal titration calorimetry, to note that the isoenzymes generally exhibit similar behavior, with calcium having only a minor effect. However, the spatial and temporal changes in the gene expression profiles of the three AtGDH genes point to AtGDH2 as the most prevalent isoform.

Protein–Protein Interfaces as Druggable Targets: A Common Motif of the Pyridoxal-5′-Phosphate-Dependent Enzymes to Receive the Coenzyme from Its Producers

Pyridoxal-5′-phosphate (PLP), a phosphorylated form of vitamin B6, acts as a coenzyme for numerous reactions, including those changed in cancer and/or associated with the disease prognosis. Since highly reactive PLP can modify cellular proteins, it is hypothesized to be directly transferred from its donors to acceptors. Our goal is to validate the hypothesis by finding common motif(s) in the multitude of PLP-dependent enzymes for binding the limited number of PLP donors, namely pyridoxal kinase (PdxK), pyridox(am)in-5′-phosphate oxidase (PNPO), and PLP-binding protein (PLPBP). Experimentally confirmed interactions between the PLP donors and acceptors reveal that PdxK and PNPO interact with the most abundant PLP acceptors belonging to structural folds I and II, while PLPBP – with those belonging to folds III and V. Aligning sequences and 3D structures of the identified interactors of PdxK and PNPO, we have identified a common motif in the PLP-dependent enzymes of folds I and II. The motif extends from the enzyme surface to the neighborhood of the PLP binding site, represented by an exposed alfa-helix, a partially buried beta-strand, and residual loops. Pathogenicity of mutations in the human PLP-dependent enzymes within or in the vicinity of the motif, but outside of the active sites, supports functional significance of the motif that may provide an interface for the direct transfer of PLP from the sites of its synthesis to those of coenzyme binding. The enzyme-specific amino acid residues of the common motif may be useful to develop selective inhibitors blocking PLP delivery to the PLP-dependent enzymes critical for proliferation of malignant cells.

Predictive significance of PRR11 in prognosis and immune infiltration of glioma patients.

Glioma, characterized by invasive growth and high recurrence, is the main cause of brain-tumor-related deaths. Currently, proline rich 11 (PRR11) has served as a reliable predictor in diagnosis, prognosis, and immunotherapeutic response of several human cancers. However, the predictive significance of PRR11 in prognosis and immune infiltration of glioma patients is still uncertain. Therefore, glioma-related information from multiple data sets and single-cell sequencing (scRNA-seq), including CGGA, TCGA_GTEx, GEO, 10x Genomics, and Smart-seq, was included in this study. Then, clinical correlation analysis, univariate and multivariate Cox analysis, Kaplan-Meier survival analysis, and receiver operating characteristic curve analysis were utilized to explore diagnostic and prognostic value of PRR11 in glioma. Besides, functional mechanisms of PRR11 in glioma were also carried out via Spearman's correlation, immune infiltration, scRNA-seq, gene ontology enrichment, and Kyoto encyclopedia of genes and genomes analysis. Finally, we selected two glioma cell lines, U251 and LN229, for validation of oncogenic role of PRR11 in glioma. As a result, PRR11 is a promising and reliable predictor in diagnosis and prognosis of glioma patients, positively correlates with clinical malignancy, World Health Organization grades, IDH mutation status, 1p19q codeletion status, histology, and poor survival time. Mechanically, PRR11 is specifically overexpressed in Mano/Macro_C21 and CD8Tex cells and interferes with cell-cycle, transcription factors, immune infiltration, MYC targets, and PI3K/AKT/mTOR signaling. Further exploration of biological mechanisms of PRR11 in glioma patients reveals that PRR11 might exert its oncogenic effects via involvement with chromosome segregation, coenzyme binding, cell cycle, and pyruvate metabolism. Moreover, PRR11 knockdown suppressed cell viability, migration, cell-cycle progression, and induced apoptosis and autophagy in glioma cells. In summary, our results suggest that PRR11 might be a novel and reliable diagnostic and prognostic predictor in glioma patients, providing a promising clinical therapeutic target for glioma.

Enzymatic Characterization and Coenzyme Specificity Conversion of a Novel Dimeric Malate Dehydrogenase from Bacillus subtilis.

Malate is an important material to various industrials and clinical applications. Bacillus subtilis is a widely used biocatalyst tool for chemical production. However, the specific enzymaticproperties of malate dehydrogenase from Bacillus subtilis (BsMDH) remain largely unknown. In the present study, BsMDH was cloned, recombinantly expressed and purified to test its enzymatic properties. The molecular weight of single unit of BsMDH was 34,869.7 Da. Matrix-Assisted Laser-Desorption Ionization-Time-of-Flight Mass Spectrometry and gel filtration analysis indicated that the recombinant BsMDH could form dimers. The kcat/Km values of oxaloacetateand NADH were higher than those of malate and NAD+, respectively, indicating a better catalysis in the direction of malate synthesis than the reverse. Furthermore, six BsMDH mutants were constructed with the substitution of amino acids at the coenzyme binding site. Among them, BsMDH-T7 showed a greatly higher affinity and catalysis efficiency to NADPH than NADH with the degree of alteration of 2039, suggesting the shift of the coenzyme dependence from NADH to NADPH. In addition, BsMDH-T7 showed a relatively lower Km value, but a higher kcat and kcat/Km than NADPH-dependent MDHs from Thermus flavus and Corynebacterium glutamicum. Overall, these results indicated that BsMDH and BsMDH-T7 mutant might be promising enzymes for malate production.

Effect of naturally-occurring mutations on the stability and function of cancer-associated NQO1: Comparison of experiments and computation.

Recent advances in DNA sequencing technologies are revealing a large individual variability of the human genome. Our capacity to establish genotype-phenotype correlations in such large-scale is, however, limited. This task is particularly challenging due to the multifunctional nature of many proteins. Here we describe an extensive analysis of the stability and function of naturally-occurring variants (found in the COSMIC and gnomAD databases) of the cancer-associated human NAD(P)H:quinone oxidoreductase 1 (NQO1). First, we performed in silico saturation mutagenesis studies (>5,000 substitutions) aimed to identify regions in NQO1 important for stability and function. We then experimentally characterized twenty-two naturally-occurring variants in terms of protein levels during bacterial expression, solubility, thermal stability, and coenzyme binding. These studies showed a good overall correlation between experimental analysis and computational predictions; also the magnitude of the effects of the substitutions are similarly distributed in variants from the COSMIC and gnomAD databases. Outliers in these experimental-computational genotype-phenotype correlations remain, and we discuss these on the grounds and limitations of our approaches. Our work represents a further step to characterize the mutational landscape of NQO1 in the human genome and may help to improve high-throughput in silico tools for genotype-phenotype correlations in this multifunctional protein associated with disease.

Transcriptomic and metabolomic analyses reveal the antifungal mechanism of the compound phenazine-1-carboxamide on Rhizoctonia solani AG1IA.

To explore the molecular mechanisms of the antifungal compound phenazine-1-carboxamide (PCN) inhibits Rhizoctonia solani and discover potential targets of action, we performed an integrated analysis of transcriptome and metabolome in R. solani mycelium by whether PCN treating or not. A total of 511 differentially expressed genes (DEGs) were identified between the PCN treatment and control groups. The fluorescence-based quantitative PCR (qPCR) got the accordant results of the gene expression trends for ten randomly selected DEGs. The Gene Ontology (GO) enrichment analysis revealed that fatty acid metabolic process, fatty acid oxidation, and lipid oxidation were among the most enriched in the biological process category, while integral component of membrane, plasma membrane, and extracellular region were among the most enriched in the cellular component category and oxidoreductase activity, cofactor binding, and coenzyme binding were among the most enriched in the molecular function category. KEGG enrichment analysis revealed the most prominently enriched metabolic pathways included ATP-binding cassette (ABC) transporters, nitrogen metabolism, aminobenzoate degradation. The DEGs related functions of cellular structures, cell membrane functions, cellular nutrition, vacuole-mitochondrion membrane contact site and ATPase activity, pH, anti-oxidation, were downregulated. A total of 466 differential metabolites were found between the PCN treatment and control groups after PCN treatment. KEGG enrichment found purine, arachidonic acid, and phenylpropanoid biosynthesis pathways were mainly affected. Further results proved PCN decreased the mycelial biomass and protein content of R. solani, and superoxide dismutase (SOD) activity reduced while peroxidase (POD) and cytochrome P450 activities increased. The molecule docking indicted that NADPH nitrite reductase, ATP-binding cassette transporter, alpha/beta hydrolase family domain-containing protein, and NADPH-cytochrome P450 reductase maybe the particular target of PCN. In conclusion, the mechanisms via which PCN inhibits R. solani AG1IA may be related to cell wall damage, cell membrane impairment, intracellular nutrient imbalance, disturbed antioxidant system, and altered intracellular pH, which laid foundation for the further new compound designing to improve antifungal efficacy.

Dependence of crystallographic atomic displacement parameters on temperature (25-150 K) for complexes of horse liver alcohol dehydrogenase.

Enzymes catalyze reactions by binding and orienting substrates with dynamic interactions. Horse liver alcohol dehydrogenase catalyzes hydrogen transfer with quantum-mechanical tunneling that involves fast motions in the active site. The structures and B factors of ternary complexes of the enzyme with NAD+ and 2,3,4,5,6-pentafluorobenzyl alcohol or NAD+ and 2,2,2-trifluoroethanol were determined to 1.1-1.3 Å resolution below the `glassy transition' in order to extract information about the temperature-dependent harmonic motions, which are reflected in the crystallographic B factors. The refinement statistics and structures are essentially the same for each structure at all temperatures. The B factors were corrected for a small amount of radiation decay. The overall B factors for the complexes are similar (13-16 Å2) over the range 25-100 K, but increase somewhat at 150 K. Applying TLS refinement to remove the contribution of pseudo-rigid-body displacements of coenzyme binding and catalytic domains provided residual B factors of 7-10 Å2 for the overall complexes and of 5-10 Å2 for C4N of NAD+ and the methylene carbon of the alcohols. These residual B factors have a very small dependence on temperature and include local harmonic motions and apparently contributions from other sources. Structures at 100 K show complexes that are poised for hydrogen transfer, which involves atomic displacements of ∼0.3 Å and is compatible with the motions estimated from the residual B factors and molecular-dynamics simulations. At 298 K local conformational changes are also involved in catalysis, as enzymes with substitutions of amino acids in the substrate-binding site have similar positions of NAD+ and pentafluorobenzyl alcohol and similar residual B factors, but differ by tenfold in the rate constants for hydride transfer.

Enhancing Acetophenone Tolerance of Anti-Prelog Short-Chain Dehydrogenase/Reductase EbSDR8 Using a Whole-Cell Catalyst by Directed Evolution

The short-chain dehydrogenase/reductase (SDR) from Empedobacter brevis ZJUY-1401 (EbSDR8, GenBank: ALZ42979.1) is a promising biocatalyst for the reduction of acetophenone to (R)-1-phenylethanol, but its industrial application is restricted by its insufficient tolerance to acetophenone. In this paper, we developed a chromogenic reaction-based high-throughput screening method and employed directed evolution to enhance the acetophenone tolerance of EbSDR8. The resulting variant, M190V, showed 74.8% improvement over the wild-type in specific activity when catalyzing the reduction of 200 mM acetophenone. Kinetic analysis revealed a 70% enhancement in its catalytic efficiency (kcat/Km). Molecular docking was conducted to reveal the possible mechanism behind the improved acetophenone tolerance, and the result implied that the M190V mutation is conducive to the binding and release of coenzyme. Aside from the improved catalytic performance when dealing with a high concentration of acetophenone, other features of M190V, such as a broad pH range (6.0 to 10.5), low optimal cosubstrate concentration (1% isopropanol), and a temperature optimum close to that of E. coli cells (35 °C), also contribute to its practical application as a whole-cell catalyst. In this study, we first designed a directed evolution means to engineer the enzyme and obtained the positive variant which has a high activity under high concentrations of acetophenone. After that, we optimized the catalytic performance of the variant to adapt to industrial applications.

Aldehyde dehydrogenase 2 and arrhythmogenesis.

Cardiac arrhythmia is a common cardiovascular disease that leads to considerable economic burdens and significant global public health challenges. Despite the remarkable progress made in recent decades, antiarrhythmic therapy remains suboptimal. Aldehyde dehydrogenase 2 (ALDH2), a critical detoxifying enzyme, catalyzes toxic aldehydes and protects individuals from damage caused by oxidative stress. Accumulating evidence has demonstrated that ALDH2 activation has potential antiarrhythmic benefits. The correlation between ALDH2 deficiency and arrhythmogenesis has been widely recognized. In this review, we summarize recent research on the potential role of ALDH2 activation and antiarrhythmic protection, as well as the role played by the ALDH2∗2 polymorphism (rs671) in promoting arrhythmic risk. Additionally, we discuss important new findings illustrating the use of ALDH2 activators, which may prove to be promising antiarrhythmic therapy agents.

Mechanism and Molecular Targets of Ejiao Siwu Decoction for Treating Primary Immune Thrombocytopenia Based on High-Performance Liquid Chromatograph, Network Pharmacology, Molecular Docking and Cytokines Validation.

We explored the mechanisms and molecular targets of Ejiao Siwu Decoction (EJSW) for treating primary immune thrombocytopenia (ITP) using network pharmacology and molecular docking. Active compounds of EJSW were identified by high-performance liquid chromatography-diode array detector (HPLC-DAD) and high-performance liquid chromatography-mass spectrometry (HPLC-MS) and their targets were obtained from HERB and SwissTargetPrediction, and ITP targets were obtained from Comparative Toxicogenomics Database (CTD) and GeneCards. STRING and Cytoscape were used for protein-protein interaction (PPI) network analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses by WebGestalt yielded a gene-pathway network, Autodock molecular docking was applied to screen targets and active compounds, and cytokines were detected using a cytometric bead array (CBA) human inflammation kit. We identified 14 compounds and 129 targets, and 1,726 ITP targets. RAC-alpha serine/threonine-protein kinase (AKT1), tumour necrosis factor (TNF), interleukin-6 (IL6), caspase-3 (CASP3) and tumour suppressor protein (TP53) were core targets (nodes and edges). Functional annotation identified cofactor binding and coenzyme binding, and 20 significantly enriched pathways. Active compounds of EJSW were successfully docked with ITP targets. Tumour necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) were upregulated in ITP patients, vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor D (VEGF-D) were downregulated, and EJSW treatment reversed these trends. EJSW may regulate key ITP targets based on the in silico analyses, and protect vascular integrity through AGE-RAGE signalling, complement and coagulation cascades, and VEGF signalling by downregulating TNF-α, IL-1β and other inflammatory factors.

BADH-NAD+-K+ Complex Interaction Studies Reveal a New Possible Mechanism between Potassium and Glutamic 254 at the Coenzyme Binding Site.

Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Incubation of porcine kidney BADH (pkBADH) with NAD+ decreases the catalytic cysteine (C288) reactivity. Potassium ion increases the pkBADH affinity by the coenzyme. This work aimed to analyze pkBADH and NAD+ interaction in the presence and absence of K+ using 1H NMR to identify the amino acids that interact with NAD+ and/or K+ to understand the regulation process of pkBADH-NAD+ complex formation mediated by the K+ ion and their impact on the substrate binding and catalysis. Nuclear magnetic resonance spectra of pkBADH were obtained in the presence and absence of NAD+ and K+. The results show a chemical shift of the signals corresponding to the catalytic glutamic that participates in the transfer of H+ in the reaction of the pkBADH-NAD+-K+ complex formation. Furthermore, there is a widening of the signal that belongs to the catalytic cysteine indicating higher rigidity or less grade of rotation of the structure, which is consistent with the possible conformations of C288 in the catalytic process; in addition, there is evidence of changes in the chemical environment that surrounds NAD+.

Structural Rearrangements of a Dodecameric Ketol-Acid Reductoisomerase Isolated from a Marine Thermophilic Methanogen.

Ketol-acid reductoisomerase (KARI) orchestrates the biosynthesis of branched-chain amino acids, an elementary reaction in prototrophic organisms as well as a valuable process in biotechnology. Bacterial KARIs belonging to class I organise as dimers or dodecamers and were intensively studied to understand their remarkable specificity towards NADH or NADPH, but also to develop antibiotics. Here, we present the first structural study on a KARI natively isolated from a methanogenic archaea. The dodecameric structure of 0.44-MDa was obtained in two different conformations, an open and close state refined to a resolution of 2.2-Å and 2.1-Å, respectively. These structures illustrate the conformational movement required for substrate and coenzyme binding. While the close state presents the complete NADP bound in front of a partially occupied Mg2+-site, the Mg2+-free open state contains a tartrate at the nicotinamide location and a bound NADP with the adenine-nicotinamide protruding out of the active site. Structural comparisons show a very high conservation of the active site environment and detailed analyses point towards few specific residues required for the dodecamerisation. These residues are not conserved in other dodecameric KARIs that stabilise their trimeric interface differently, suggesting that dodecamerisation, the cellular role of which is still unknown, might have occurred several times in the evolution of KARIs.

Transcriptome-Based Network Analysis Reveals Hirudin Potentiates Anti-Renal Fibrosis Efficacy in UUO Rats.

Background: Hirudin has been widely used in the treatment of antifibrosis. Previous studies have shown that hirudin can effectively improve the clinical remission rate of chronic kidney disease. However, the mechanism of its renal protection has not been systematically investigated.Methods: In this study, the reliability of UUO-induced renal interstitial fibrosis was evaluated by histopathological verification. High-throughput transcriptome sequencing was used to elucidate the molecular mechanism of hirudin, differentially expressed mRNAs were identified, and their functions were analyzed by GO analysis and GSEA. In addition, the RNA-seq results were validated by in vitro and vivo experiments.Results: We found 322 identical differential expressed genes (IDEs) in the UUO hirudin-treated group compared with the sham group. Functional enrichment analysis indicated that cellular amino acid metabolic processes were the most obvious enrichment pathways in biological processes. In terms of molecular functional enrichment analysis, IDEs were mainly enriched in coenzyme binding, pyridoxal phosphate binding and other pathways. In addition, microbody is the most obvious pathway for cellular components. A total of 115 signaling pathways were enriched, and AMPK, JAK-STAT, and PI3K-Akt signaling pathways were the important signaling pathways enriched. We found that PI3K, p-Akt, and mTOR expression were significantly reduced by hirudin treatment. In particular, our results showed that hirudin could induce a decrease in the expression of autophagy-related proteins such as P62, LC3, Beclin-1 in TGF-β1-induced NRK-52E cells.Conclusion: Our results suggest that hirudin may protect the kidney by ameliorating renal autophagy impairment through modulating the PI3K/Akt pathway.