Substrates For Reductase Research Articles

Synthesis of Caged HMG-CoA Reductase Substrates for the Elucidation of Cellular Pathways.

The synthesis of photocaged substrates of the biologically important enzyme HMG-CoA reductase is reported. HMG-CoA bearing a p-hydroxyphenacyl (pHP) photocage moiety was synthesized in an overall yield of 14% over seven steps in addition to caged forms of mevalonate and mevaldehyde. The absorption maximum and quantum yield for the decaging of the photocaged compounds are dependent on pH with a λmax of 330 nm and a ϕ of 5%, respectively, at pH 9.1 but a λmax of 290 nm and a ϕ of 16%, respectively, at pH 6.7.

Dietary- and host-derived metabolites are used by diverse gut bacteria for anaerobic respiration.

Respiratory reductases enable microorganisms to use molecules present in anaerobic ecosystems as energy-generating respiratory electron acceptors. Here we identify three taxonomically distinct families of human gut bacteria (Burkholderiaceae, Eggerthellaceae and Erysipelotrichaceae) that encode large arsenals of tens to hundreds of respiratory-like reductases per genome. Screening species from each family (Sutterella wadsworthensis, Eggerthella lenta and Holdemania filiformis), we discover 22 metabolites used as respiratory electron acceptors in a species-specific manner. Identified reactions transform multiple classes of dietary- and host-derived metabolites, including bioactive molecules resveratrol and itaconate. Products of identified respiratory metabolisms highlight poorly characterized compounds, such as the itaconate-derived 2-methylsuccinate. Reductase substrate profiling defines enzyme-substrate pairs and reveals a complex picture of reductase evolution, providing evidence that reductases with specificities for related cinnamate substrates independently emerged at least four times. These studies thus establish an exceptionally versatile form of anaerobic respiration that directly links microbial energy metabolism to the gut metabolome.

Исследование активности глутатионредуктазы в коре головного мозга, стволе мозга и мозжечке облучённых крыс

The paper presents results of the study of pressing problems related to molecular mechanisms of radiation effects on a human body, as well as radiation protection and safety control to human beings in healthcare settings. Results of research on activity of glutathione reductase, the enzyme of the antioxidant system, in various structural-functional brain areas in the white outbred rats exposed to whole-body gamma radiation at a dose of 6 Gy are considered. Glutathione reductase activity was found in the cerebral cortex, brain stem and cerebellum of irradiated animals. The activity of glutathione reductase in the cytoplasm was higher than in mitochondria derived from cortex, cerebellum and brain stem cells in the intact animals. The sharp decrease in glutathione reductase activity was observed on the 7th day after the exposure of rats to gamma radiation. The activity of glutathione reductase in the studied brain areas of irradiated rats changes with the time of radiation lesion development. The role of the enzyme in the metabolism of glutathione, a substrate of glutathione reductase, in rats brain undergone acute whole-body radiation injury, is discussed.

The regulatory mechanisms of proline and hydroxyproline metabolism: Recent advances in perspective.

For diverse human tumors, growth and metastasis are dependent on proline synthesis, but the mechanisms underlying this association are not clear. Proline incorporated into collagen is primarily synthesized from glutamine. Thus, rates of collagen synthesis are modulated by the enzymes of proline synthesis. On the other hand, the hydroxylation of collagen proline requires αKG, ascorbate and ferrous iron, substrates necessary for the epigenetic demethylation of DNA and histones. The metabolic relationship of proline and hydroxyproline degradation are initiated by distinct dehydrogenases but the respective oxidized products, P5C and OH-P5C are substrates for P5C Reductase and P5C Dehydrogenase allowing for mutual competition. This provides a model by which proline synthesis in cancer plays a role in reprogramming gene expression. The metabolism of proline and hydroxyproline are also linked to the HIF response to hypoxia. Hypoxia increased the expression of ALDH18A1, which is the limiting step in proline and collagen synthesis. Hydroxyproline increases levels of HIF-1α presumably by inhibiting its degradation. These new findings allow the suggestion that there is a regulatory axis from glutamine to proline and collagen synthesis, and the release of free hydroxyproline can feed back on the HIF pathway.

Transcriptome and Metabolome Profiling Reveal the Resistance Mechanisms of Rice against Brown Planthopper.

Brown planthopper (Nilaparvata lugens Stål, BPH) is one of the most destructive insects affecting rice production. To better understand the physiological mechanisms of how rice responds to BPH feeding, we analyzed BPH-induced transcriptomic and metabolic changes in leaf sheaths of both BPH-susceptible and -resistant rice varieties. Our results demonstrated that the resistant rice reduced the settling, feeding and growth of BPH. Metabolic analyses indicated that BPH infestation caused more drastic overall metabolic changes in the susceptible variety than the resistant rice. Differently accumulated metabolites (DAMs) belonging to flavonoids were downregulated in the susceptible rice but upregulated in resistant variety. Transcriptomic analyses revealed more differentially expressed genes (DEGs) in susceptible rice than resistant rice, and DEGs related to stimulus were significantly upregulated in resistant rice but downregulated in susceptible rice. Combined analyses of transcriptome and metabolome showed that many DEGs and DAMs were enriched in phenylpropane biosynthesis, flavonoid biosynthesis, and plant hormone signal transduction. We conducted correlation analyses of DEGs and DAMs in these pathways and found a high correlation between DEGs and DAMs. Then, we found that the contents of endogenous indole 3-acetic acid (IAA) in resistant rice was lower than that of susceptible rice after BPH feeding, while the salicylic acid (SA) content was the opposite. For functional analysis, an exogenous application of IAA decreased rice resistance to BPH, but the exogenous application of SA increased resistance. In addition, biochemical assessment and quantitative PCR analysis showed that the lignin content of resistant accession was constitutively higher than in susceptible accession. By adding epigallocatechin, the substrate of anthocyanidin reductase (ANR), to the artificial diet decreased the performance of BPH. We first combined a transcriptome-metabolome-wide association study (TMWAS) on rice resistance to BPH in this study. We demonstrated that rice promoted resistance to BPH by inducing epigallocatechin and decreasing IAA. These findings provided useful transcriptomic and metabolic information for understanding the rice-BPH interactions.

Emodin inhibited NADPH-quinone reductase competitively and induced cytotoxicity in rat primary hepatocytes

Consumption of Cassia occidentalis (CO) seeds, a ubiquitously distributed weed plant, is responsible for a pathological condition known as hepato-myo-encephalopathy (HME). The toxicity of CO seeds is largely attributed to the presence of anthraquinones (AQs). Here, we report that Emodin, a CO anthraquinone, inhibits the enzymatic activity of NADPH-Quinone reductase, which is an intracellular enzyme fundamentally involved in the detoxification of quinone containing compounds. Emodin binds to the active site of the enzyme and acts as a competitive inhibitor with respect to 2, 6-Dichlorophenolindophenol, a known substrate of NADPH-Quinone reductase. Moreover, our in-vitro study further revealed that Emodin was cytotoxic to primary rat hepatocytes.

Chlamydomonas reinhardtii, an Algal Model in the Nitrogen Cycle.

Nitrogen (N) is an essential constituent of all living organisms and the main limiting macronutrient. Even when dinitrogen gas is the most abundant form of N, it can only be used by fixing bacteria but is inaccessible to most organisms, algae among them. Algae preferentially use ammonium (NH4+) and nitrate (NO3−) for growth, and the reactions for their conversion into amino acids (N assimilation) constitute an important part of the nitrogen cycle by primary producers. Recently, it was claimed that algae are also involved in denitrification, because of the production of nitric oxide (NO), a signal molecule, which is also a substrate of NO reductases to produce nitrous oxide (N2O), a potent greenhouse gas. This review is focused on the microalga Chlamydomonas reinhardtii as an algal model and its participation in different reactions of the N cycle. Emphasis will be paid to new actors, such as putative genes involved in NO and N2O production and their occurrence in other algae genomes. Furthermore, algae/bacteria mutualism will be considered in terms of expanding the N cycle to ammonification and N fixation, which are based on the exchange of carbon and nitrogen between the two organisms.

2,2'-Dipyridyl diselenide (Py2Se2) induces G1 arrest and apoptosis in human lung carcinoma (A549) cells through ROS scavenging and reductive stress.

Organo-diselenides are well documented for pro-oxidant effects in tumor cells. However, the present study demonstrated that 2,2'-dipyridyl diselenide (Py2Se2) induced cytotoxicity in human non-small cell lung carcinoma (A549) cells through reductive stress marked by a significant decrease in the basal level of reactive oxygen species and a concurrent decrease in the ratio of oxidised (GSSG) and reduced (GSH) glutathione. The IC50 (concentration inducing 50% cytotoxicity) of Py2Se2 in A549 and human normal lung fibroblast (WI38) cells was ∼8.5 μM and ∼5.5 μM, respectively, indicating that Py2Se2 did not exhibit selective toxicity towards cancer cells. Cell free studies indicated that Py2Se2 acted as a substrate of thioredoxin reductase (TrxR) and accordingly it was proposed that TrxR mediated reduction of Py2Se2 within cells might be generating intermediates leading to a reductive environment. Despite generating a reducing environment, Py2Se2 caused significant DNA damage, G1 phase arrest and apoptosis. The mechanistic investigation revealed that Py2Se2 induced G1 arrest was mediated through up-regulation of p21 transcript in a p53 independent manner. Further, the apoptotic effect of Py2Se2 was associated with the increase in the levels of unfolded protein response markers like BiP and CHOP, mitochondrial permeability (JC1) and apoptotic markers such as cleaved caspase-3 and poly (ADP-ribose) polymerase. Finally, pre-treatment with N-acetylcysteine (a stimulator of GSH biosynthesis) or l-buthionine sulfoximine (an inhibitor of GSH biosynthesis) increased and decreased the Py2Se2 mediated apoptosis, respectively. This confirmed that the cytotoxicity of Py2Se2 in A549 cells was triggered through reductive stress.

The Functions of Chloroplast Glutamyl-tRNA in Translation and Tetrapyrrole Biosynthesis.

The chloroplast glutamyl-tRNA (tRNAGlu) is unique in that it has two entirely different functions. In addition to acting in translation, it serves as the substrate of glutamyl-tRNA reductase (GluTR), the enzyme catalyzing the committed step in the tetrapyrrole biosynthetic pathway. How the tRNAGlu pool is distributed between the two pathways and whether tRNAGlu allocation limits tetrapyrrole biosynthesis and/or protein biosynthesis remains poorly understood. We generated a series of transplastomic tobacco (Nicotiana tabacum) plants to alter tRNAGlu expression levels and introduced a point mutation into the plastid trnE gene, which has been reported to uncouple protein biosynthesis from tetrapyrrole biosynthesis in chloroplasts of the protist Euglena gracilis We show that, rather than comparable uncoupling of the two pathways, the trnE mutation is lethal in tobacco because it inhibits tRNA processing, thus preventing translation of Glu codons. Ectopic expression of the mutated trnE gene uncovered an unexpected inhibition of glutamyl-tRNA reductase by immature tRNAGlu We further demonstrate that whereas overexpression of tRNAGlu does not affect tetrapyrrole biosynthesis, reduction of GluTR activity through inhibition by tRNAGlu precursors causes tetrapyrrole synthesis to become limiting in early plant development when active photosystem biogenesis provokes a high demand for de novo chlorophyll biosynthesis. Taken together, our findings provide insight into the roles of tRNAGlu at the intersection of protein biosynthesis and tetrapyrrole biosynthesis.

Novel Insights into Gallium's Mechanism of Therapeutic Action: A DFT/PCM Study of the Interaction between Ga3+ and Ribonucleotide Reductase Substrates.

The broadly accepted mechanism of gallium's therapeutic action postulates the inactivation of the upregulated/hyperactive enzyme ribonucleotide reductase (RNR) in cancer cells by substituting the redox-active iron by redox-silent gallium in the enzyme active site. Recently, another hypothesis for the Ga3+ curative effect has been put forward: the metal cation can deactivate the enzyme by entrapping its substrates (nucleotide diphosphates; NDPs) into Ga3+-NDP complexes, lowering the free substrate levels in the cell. Several questions arise: Does gallium readily form complexes with NDPs? What are the preferable modes of metal binding to NDPs? Does, and if so, to what extent, the metal binding alter the native conformation of the substrate, thus influencing the process of substrate-enzyme recognition? Here, by employing density functional theory (DFT)/polarizable continuum model (PCM) calculations, we attempt to answer these questions. The results, which are in line with the available experimental data, lay support to the recent hypothesis about the curative effect of gallium, revealing that, by engaging the free NDPs in forming metal complexes, on the one side, and producing metal constructs that are not/poorly recognizable by the host enzyme, on the other side, gallium deprives RNR from its substrates, thus reducing the enzyme activity in malignant cells.

Fluorogenic 7-azidocoumarin and 3/4-azidophthalimide derivatives as indicators of reductase activity in microorganisms.

A series of fluorogenic heterocyclic azides were prepared and assessed as reductase substrates across a selection of Gram-negative and Gram-positive microorganisms. The majority of these azides showed similar activity profiles to nitroreductase substrates. Microorganisms that do not produce hydrogen sulfide reduced the azides, indicating reductase activity was not linked to hydrogen sulfide production.

Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

Role of the global regulator Rex in control of NAD+‐regeneration in Clostridioides (Clostridium) difficile

For the human pathogen Clostridioides (also known as Clostridium) difficile, the ability to adapt to nutrient availability is critical for its proliferation and production of toxins during infection. Synthesis of the toxins is regulated by the availability of certain carbon sources, fermentation products and amino acids (e.g. proline, cysteine, isoleucine, leucine and valine). The effect of proline is attributable at least in part to its role as an inducer and substrate of D-proline reductase (PR), a Stickland reaction that regenerates NAD+ from NADH. Many Clostridium spp. use Stickland metabolism (co-fermentation of pairs of amino acids) to generate ATP and NAD+ . Synthesis of PR is activated by PrdR, a proline-responsive regulatory protein. Here we report that PrdR, in the presence of proline, represses other NAD+ -generating pathways, such as the glycine reductase and succinate-acetyl CoA utilization pathways leading to butyrate production, but does so indirectly by affecting the activity of Rex, a global redox-sensing regulator that responds to the NAD+ /NADH ratio. Our results indicate that PR activity is the favored mechanism for NAD+ regeneration and that both Rex and PrdR influence toxin production. Using the hamster model of C. difficile infection, we revealed the importance of PrdR-regulated Stickland metabolism in the virulence of C. difficile.

Molecular Dynamic Simulation of Hydroxymethylglutaryl-CoA Reductase Inhibitors from Gnetum gnemon L. Seed Extract

Objective: Gnetum gnemon L. (melinjo) seed extract contained trans-resveratrol which has been shown to inhibit hydroxymethylglutaryl-CoA (HMG-CoA) reductase. Therefore it has a potent activity for lowering blood cholesterol. This study was carried out to determine the molecular dynamics simulation of HMG-CoA reductase inhibitors from Gnetum gnemon L. seed extract. Methods: Molecular dynamics simulation using AMBER was used. The simulation was set at 300 K as default temperature and 310 K, average human body temperature. The main parameters of this study were ligand-residue interaction, binding affinity, root mean square deviation (RMSD), root mean square fluctuation (RMSF), hydrogen bonds analysis, molecular mechanics Poisson Boltzmann surface area (MMPBSA), and molecular mechanics generalized born surface area (MMGBSA). Results: In the simulation study, trans-resveratrol, trans-piceid, gnemonol M, gnemonoside B, viniferin and gnetin C had shown lower energy than HMG (PDB ID: MAH), the substrate of HMG-CoA Reductase. Free energy binding obtained from simulation was between 11.1 to -31.38 kcal/mol. Conclusion: The simulation at 310 K was preferable than 300 K as more interactions were performed and higher affinity was obtained.

Multi-omics analyses of red blood cell reveal antioxidation mechanisms associated with hemolytic toxicity of gossypol.

Gossypol is an antiproliferative drug with limited use due to its hemolytic toxicity. In this study, accelerated hemolysis was observed in the cows treated with gossypol. Comparative metabolomics were used to gain responsive pathways in the red blood cell (RBC) to the treatment, which were crossly validated by parallel iTRAQ-based proteomic analysis and enzyme activity assay. We found that gossypol treatment appeared to considerably activate pentose phosphate pathway (PPP) with an increased key product of ribose-5-phosphate and the increased abundance and activity of several key enzymes such as 6-phosphogluconate dehydrogenase, flavin reductase, and ribose-phosphate pyrophesphokinase. Meanwhile, a decreased glycolysis metabolism was observed, as many input metabolites of glycolysis were reduced in the gossypol group, whereas its distal metabolites were unchanged, along with decreased abundance of triosephosphate isomerase and increased abundance of enzymes catalyzing several distal glycolytic steps. Oxidative reduction pathways were also remarkably affected as we found a decreased substrate of flavin reductase, glutathione disulfide, increased glutathione reductase activity, and increased abundance and activity of glutathione S-transferase with the increase of its catalytic product, cysteine. Our results demonstrated that glycolysis, PPP, and oxidative reduction pathways of RBC were all involved in RBC’s response to the hemolytic toxicity of gossypol.

Glutathione and thioredoxin systems of the malaria parasite Plasmodium falciparum: Partners in crime?

In P. falciparum, antioxidant proteins of the glutathione and thioredoxin systems are compartmentalized. Some subcellular compartments have only a partial complement of these proteins. This lack of key anti-oxidant proteins in certain sub-cellular compartments might be compensated by functional complementation between these systems. By assessing the cross-talk between these systems, we show for the first time, that the glutathione system can reduce thioredoxins that are poor substrates for thioredoxin reductase (Thioredoxin-like protein 1 and Thioredoxin 2) and thioredoxins that lack access to thioredoxin reductase (Thioredoxin 2). Our data suggests that crosstalk between the glutathione and thioredoxin systems does exist; this could compensate for the absence of certain antioxidant proteins from key subcellular compartments.

Radiopharmaceuticals in Tumor Hypoxia Imaging: A Review Focused on Medicinal Chemistry Aspects.

Since its first description in 1955, tumor hypoxia has become a central issue in cancer treatment. Since then, it is essential to diagnose accurately the tumor oxygenation degree in order to establish the appropriate treatment. In this regard, a wide diversity of radiopharmaceuticals for in vivo imaging has been developed. Special conditions of the hypoxic microenvironment are low O2 partial pressure, enhanced levels of reductases, and genetic-adaptation-expression biomolecules involved in angiogenesis, erythropoiesis, cellular proliferation, apoptosis, metabolism- and glucose-uptake, local invasion, and metastatic spread. The development of radiolabeled hypoxia markers has been based on reductase substrates, like bioreductive ligands, or on entities capable of recognizing overexpressed proteins under hypoxia conditions, i.e. HIF-1α and carbonic anhydrase IX, among others. In this review these hypoxia markers are analyzed focusing on their medicinal chemistry characteristics.

171 - Design and Validation of Novel Fluorogenic Substrates for S-Nitrosoglutathione Reductase

171 - Design and Validation of Novel Fluorogenic Substrates for S-Nitrosoglutathione Reductase

Molecular, functional and evolutionary characterization of the gene encoding HMG-CoA reductase in the fission yeast, Schizosaccharomyces pombe.

The synthesis of mevalonate, a molecule required for both sterol and isoprene biosynthesis in eukaryotes, is catalysed by 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Using a gene dosage approach, we have isolated the gene encoding HMG-CoA reductase hmgl+, from the fission yeast Schizosaccharomyces pombe (Accession Number L76979). Specifically, hmgl+ was isolated on the basis of its ability to confer resistance to lovastatin, a competitive inhibitor of HMG-CoA reductase. Gene disruption analysis showed that hmgl+ was an essential gene. This result provided evidence that, unlike Saccharomyces cerevisiae, S. pombe contained only a single functional HMG-CoA reductase gene. The presence of a single HMG-CoA reductase gene was confirmed by genomic hybridization analysis. As observed for the S. cerevisiae HMGlp, the hmgl+ protein induced membrane proliferations known as karmellae. A previously undescribed 'feed-forward' regulation was observed in which elevated levels of HMG-CoA synthase, the enzyme catalysing the synthesis of the HMG-CoA reductase substrate, induced elevated levels of hmgl+ protein in the cell and conferred partial resistance to lovastatin. The amino acid sequences of yeast and human HMG-CoA reductase were highly divergent in the membrane domains, but were extensively conserved in the catalytic domains. We tested whether the gene duplication that produced the two functional genes in S. cerevisiae occurred before or after S. pombe and S. cerevisiae diverged by comparing the log likelihoods of trees specified by these hypotheses. We found that the tree specifying post-divergence duplication had significantly higher likelihood. Moreover, phylogenetic analyses of available HMG-CoA reductase sequences also suggested that the lineages of S. pombe and S. cerevisiae diverged approximately 420 million years ago but that the duplication event that produced two HMG-CoA reductase genes in the budding yeast occurred only approximately 56 million years ago. To date, S. pombe is the only unicellular eukaryote that has been found to contain a single HMG-CoA reductase gene. Consequently, S. pombe may provide important opportunities to study aspects of the regulation of sterol biosynthesis that have been difficult to address in other organisms and serve as a test organism to identify novel therapies for modulating cholesterol synthesis.

Nor-Mevaldic acid surrogates as selective antifungal agent leads against Botrytis cinerea. Enantioselective preparation of 4-hydroxy-6-(1-phenylethoxy)tetrahydro-2H-pyran-2-one

Solvent-free desymmetrisation of meso-dialdehyde 1 with chiral 1-phenylethan-1-ol, led to preparation of 4-silyloxy-6-alkyloxytetrahydro-2H-pyran-2-one (+)-3a with a 96:4 dr Deprotected lactone (+)-19a and the related racemic lactones 16a–18a present a lactone moiety resembling the natural substrate of HMG-CoA reductase and their antifungal properties have been evaluated against the phytopathogenic fungi Botrytis cinerea and Colletotrichum gloeosporioides. These compounds were selectively active against B. cinerea, while inactive against C. gloeosporioides.