Aldehyde oxidase 1 activity and protein expression in human, rabbit, and pig ocular tissues.

Aldehyde oxidase (AOX) is a cytosolic drug-metabolizing enzyme which has attracted increasing attention in drug development due to its high hepatic expression, broad substrate profile and species differences. In contrast, there is limited information on the presence and activity of AOX in extrahepatic tissues including ocular tissues. Because several ocular drugs are potential substrates for AOX, we performed a comprehensive analysis of the AOX1 expression and activity profile in seven ocular tissues from humans, rabbits, and pigs. AOX activities were determined using optimized assays for the established human AOX1 probe substrates 4-dimethylamino-cinnamaldehyde (DMAC) and phthalazine. Inhibition studies were undertaken in conjunctival and retinal homogenates using well-established human AOX1 inhibitors menadione and chlorpromazine. AOX1 protein contents were quantitated with targeted proteomics and confirmed by immunoblotting. Overall, DMAC oxidation rates varied over 10-fold between species (human ˃˃ rabbit ˃ pig) and showed 2- to 6-fold differences between tissues from the same species. Menadione seemed a more potent inhibitor of DMAC oxidation across species than chlorpromazine. Human AOX1 protein levels were highest in the conjunctiva, followed by most posterior tissues, whereas anterior tissues showed low levels. The rabbit AOX1 expression was high in the conjunctiva, retinal pigment epithelial (RPE), and choroid while lower in the anterior tissues. Quantification of pig AOX1 was not successful but immunoblotting confirmed the presence of AOX1 in all species. DMAC oxidation rates and AOX1 contents correlated quite well in humans and rabbits. This study provides, for the first time, insights into the ocular expression and activity of AOX1 among multiple species.

Phylloquinone is preferable over menadione as a growth factor for Porphyromonas gingivalis.

OBJECTIVES: Porphyromonas gingivalis is the etiological agent of chronic periodontitis. Menadione (vitamin K3) and phylloquinone (vitamin K1) are well-known growth factors for P. gingivalis, while menadione is widely used in growth experiments. Here we attempted to determine the differences in phylloquinone and menadione in P. gingivalis growth experiments, which have not been well studied to date. METHODS: We investigated the effects of menadione and phylloquinone on the growth of two W83 strains and seven ATCC 33277 strains of P. gingivalis. RESULTS: The ATCC 33277 strains grew well with phylloquinone at 2.9 µM in a complex medium (nutrient medium) and at 29 µM in two minimal media. In contrast, the W83 strains grew well without menadione or phylloquinone in three different culture media. Menadione at 2.9 µM, the conventionally used concentration for culturing P. gingivalis, supported the growth of most ATCC 33277 strains but inhibited the growth of some W83 and ATCC 33277 strains. Furthermore, menadione at 14.5 µM frequently inhibited cell growth, while phylloquinone at 145 µM promoted cell growth. CONCLUSIONS: These results indicate that menadione and phylloquinone act as growth factors for ATCC 33277 but that menadione also can inhibit P. gingivalis growth. Thus, we propose that phylloquinone be used instead of menadione in P. gingivalis growth experiments requiring vitamin K.

Asphyxiation of Metarhizium robertsii during mycelial growth produces conidia with increased stress tolerance via increased expression of stress-related genes.

Little is known about the impact of hypoxia and anoxia during mycelial growth on tolerance to different stress conditions of developing fungal conidia. Conidia of the insect-pathogenic fungus Metarhizium robertsii were produced on potato dextrose agar (PDA) medium under normoxia (control = normal oxygen concentrations), continuous hypoxia, and transient anoxia, as well as minimal medium under normoxia. The tolerance of the conidia produced under these different conditions was evaluated in relation to wet heat (heat stress), menadione (oxidative stress), potassium chloride (osmotic stress), UV radiation, and 4-nitroquinoline-1-oxide (=4-NQO genotoxic stress). Growth under hypoxic condition induced higher conidial tolerance of M. robertsii to menadione, KCl, and UV radiation. Transient anoxic condition induced higher conidial tolerance to KCl and UV radiation. Nutritional stress (i.e., minimal medium) induced higher conidial tolerance to heat, menadione, KCl, and UV radiation. However, neither of these treatments induced higher tolerance to 4-NQO. The gene hsp30 and hsp101 encoding a heat shock protein was upregulated under anoxic condition. In conclusion, growth under hypoxia and anoxia produced conidia with higher stress tolerances than conidia produced in normoxic condition. The nutritive stress generated by minimal medium, however, induced much higher stress tolerances. This condition also caused the highest level of gene expression in the hsp30 and hsp101 genes. Thus, the conidia produced under nutritive stress, hypoxia, and anoxia had greater adaptation to stress.

Evaluating protective effects of botanicals under inflammation and oxidative stress in chicken apical-out enteroids.

Botanicals (BOTs) are well known for their anti-inflammatory and antioxidant activities. They have been widely used as feed additives to reduce inflammation and improve intestinal functions in agricultural animals. However, the effects of BOTs on chicken intestinal epithelial functions are not fully understood. The 3D apical-out chicken enteroids recapitulate the intestinal tissue, and allow convenient access to the luminal surface, thus serving as a suitable model for investigating gut functions. The aim of this study was to identify the roles of BOTs in protecting the intestinal epithelium in chicken enteroids under challenging conditions. Apical-out enteroids were isolated from the small intestines of 18 days-old chicken embryos. Lipopolysaccharide (LPS, 10 µg/mL) and menadione (400 µM) challenges were performed in the media with or without BOTs. Paracellular Fluorescein isothiocyanate-dextran 4kD (FD4) permeability, inflammatory cytokine gene expression, and reactive oxygen species (ROS) generation were analyzed post-BOTs and challenges treatments. Statistical analysis was performed using one-way ANOVA and post hoc multiple comparisons among treatments. The results showed that the LPS challenge for 24 h induced a 50% increase in FD4 permeability compared with nontreated control; thymol, thyme essential oil, and phenol-rich extract significantly (P < 0.02) reduced FD4 permeability by 25%, 41%, and 48% respectively, in comparison with LPS treatment. Moreover, the gene expression of inflammatory cytokines was upregulated, tight junction proteins and defensins were downregulated (P < 0.05) after 6 h of LPS treatment, while these BOTs treatments significantly restored the LPS-induced gene expression alterations (P < 0.05). Menadione oxidative challenge for 1 h significantly increased the ROS level compared with unchallenged control. Enteroids treated with thymol and thyme essential oils showed 30% reduced ROS levels, while the phenol-rich extract reduced them by 60%, in comparison with the challenged group (P < 0.0001). These data confirmed the role of BOTs in supporting the barrier function and reducing the disruptive effects of inflammation and oxidation in the chicken intestine.

Development of a model for studying the developmental consequences of oxidative sperm DNA damage by targeting redox-cycling naphthoquinones to the Sertoli cell population.

Oxidative stress can be induced in the testes by a wide range of factors, including scrotal hyperthermia, varicocele, environmental toxicants, obesity and infection. The clinical consequences of such stress include the induction of genetic damage in the male germ line which may, in turn, have serious implications for the health and wellbeing of the progeny. In order to confirm the transgenerational impact of oxidative stress in the testes, we sought to develop an animal model in which this process could be analysed. Our primary approach to this problem was to induce Sertoli cells (robust, terminally differentiated, tissue-specific testicular cells whose radioresistance indicates significant resistance to oxidative stress) to generate high levels of reactive oxygen species (ROS) within the testes. To achieve this aim, six follicle-stimulating hormone (FSH) peptides were developed and compared for selective targeting to Sertoli cells both in vitro and in vivo. Menadione, a redox-cycling agent, was then conjugated to the most promising FSH candidate using a linker that had been optimised to enable maximum production of ROS in the targeted cells. A TM4 Sertoli cell line co-incubated with the FSH-menadione conjugate in vitro exhibited significantly higher levels of mitochondrial ROS generation (10-fold), lipid peroxidation (2-fold) and oxidative DNA damage (2-fold) than the vehicle control. Additionally, in a proof-of-concept study, ten weeks after a single injection of the FSH-menadione conjugate in vivo, injected male mice were found to exhibit a 1.6 fold increase in DNA double strand breaks and 13-fold increase in oxidative DNA damage to their spermatozoa while still retaining their ability to initiate a pregnancy. We suggest this model could now be used to study the influence of chronic oxidative stress on testicular function with emphasis on the impact of DNA damage in the male germ line on the mutational profile and health of future generations.

The conditional mitochondrial protein complexome in the Arabidopsis thaliana root and shoot.

Protein complexes are important for almost all biological processes. Hence, to fully understand how cells work, it is also necessary to characterize protein complexes and their dynamics in response to various cellular cues. Moreover, the dynamics of protein interaction play crucial roles in regulating the (dis)association of protein complexes and, in turn, regulating biological processes such as metabolism. Here, mitochondrial protein complexes were investigated by blue native PAGE and size-exclusion chromatography under conditions of oxidative stress in order to monitor their dynamic (dis)associations. Rearrangements of enzyme interactions and changes in protein complex abundance were observed in response to oxidative stress induced by menadione treatment. These included changes in enzymatic protein complexes involving γ-amino butyric acid transaminase (GABA-T), Δ-ornithine aminotransferase (Δ-OAT), or proline dehydrogenase 1 (POX1) that are expected to affect proline metabolism. Menadione treatment also affected interactions between several enzymes of the tricarboxylic acid (TCA) cycle and the abundance of complexes of the oxidative phosphorylation pathway. In addition, we compared the mitochondrial complexes of roots and shoots. Considerable differences between the two tissues were observed in the mitochondrial import/export apparatus, the formation of super-complexes in the oxidative phosphorylation pathway, and specific interactions between enzymes of the TCA cycle that we postulate may be related to the metabolic/energetic requirements of roots and shoots.

Age and oxidative stress regulate Nrf2 homeostasis in human articular chondrocytes.

OBJECTIVE: The purpose of this study was to investigate the effect of age and oxidative stress on regulation of nuclear factor erythroid-2-related factor 2 (Nrf2) in young, old, and osteoarthritic (OA) human articular chondrocytes. DESIGN: Levels of Nrf2 in primary human chondrocytes isolated from young, old, and OA donors were measured by immunoblotting, qPCR, and immunohistochemistry. Effects on levels of Nrf2, antioxidant proteins regulated by Nrf2, as well as p65, and the anabolic response to insulin-like growth factor-1 (IGF-1) were evaluated after induction of oxidative stress with menadione, Nrf2 knockdown with siRNA, and/or Nrf2 activation with RTA-408. RESULTS: Nrf2 protein levels were significantly lower in older adult chondrocytes (∼0.59 fold; p = 0.034) and OA chondrocytes (∼0.50 fold; p = 0.016) compared to younger cells. Menadione significantly increased Nrf2 protein levels in young chondrocytes by just under four-fold without changes in old chondrocytes. Nrf2 knockdown and activation differentially regulated levels of anti-oxidant proteins including sulfiredoxin and NAD(P)H quinone dehydrogenase 1. Nrf2 activation with RTA-408 also decreased basal p65 phosphorylation, increased aggrecan and type II collagen gene expression, and increased production of proteoglycans in OA chondrocytes treated with IGF-1. CONCLUSIONS: Targeted therapeutic strategies aimed at maintaining Nrf2 activity could be useful in maintaining chondrocyte homeostasis through maintenance of intracellular antioxidant function and redox balance.

Vitamin K Contribution to DNA Damage-Advantage or Disadvantage? A Human Health Response.

Vitamin K is the common name for a group of compounds recognized as essential for blood clotting. The group comprises phylloquinone (K1)-a 2-methyl-3-phytyl-1,4-naphthoquinone; menaquinone (K2, MK)-a group of compounds with an unsaturated side chain in position 3 of a different number of isoprene units and a 1,4-naphthoquinone group and menadione (K3, MD)-a group of synthetic, water-soluble compounds 2-methyl-1,4-naphthoquinone. However, recent epidemiological studies suggest that vitamin K has various benefits that go beyond blood coagulation processes. A dietary intake of K1 is inversely associated with the risk of pancreatic cancer, K2 has the potential to induce a differentiation in leukemia cells or apoptosis of various types of cancer cells, and K3 has a documented anti-cancer effect. A healthy diet rich in fruit and vegetables ensures an optimal supply of K1 and K2, though consumers often prefer supplements. Interestingly, the synthetic form of vitamin K-menadione-appears in the cell during the metabolism of phylloquinone and is a precursor of MK-4, a form of vitamin K2 inaccessible in food. With this in mind, the purpose of this review is to emphasize the importance of vitamin K as a micronutrient, which not only has a beneficial effect on blood clotting and the skeleton, but also reduces the risk of cancer and other pro-inflammatory diseases. A proper diet should be a basic and common preventive procedure, resulting in a healthier society and reduced burden on healthcare systems.

Vitamin K3 inhibits FtsZ assembly, disrupts the Z-ring in Streptococcus pneumoniae and displays anti-pneumococcal activity.

The respiratory pathogen, Streptococcus pneumoniae has acquired multiple-drug resistance over the years. An attractive strategy to combat pneumococcal infection is to target cell division to inhibit the proliferation of S. pneumoniae. This work presents Vitamin K3 as a potential anti-pneumococcal drug that targets FtsZ, the master coordinator of bacterial cell division. Vitamin K3 strongly inhibited S. pneumoniae proliferation with a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) of 6 µg/ml. Vitamin K3 disrupted the Z-ring localization in both S. pneumoniae and Bacillus subtilis within 30 min of treatment, while the membrane integrity and nucleoid segregation remain unchanged. Several complementary experiments showed that Vitamin K3 inhibits the assembly of purified S. pneumoniae FtsZ (SpnFtsZ) and induces conformational changes in the protein. Interestingly, Vitamin K3 interfered with GTP binding onto FtsZ and increased the GTPase activity of FtsZ polymers. The intrinsic tryptophan fluorescence of SpnFtsZ revealed that Vitamin K3 delays the nucleation of FtsZ polymers and reduces the rate of polymerization. In the presence of a non-hydrolyzable analog of GTP, Vitamin K3 did not show inhibition of FtsZ polymerization. These results indicated that Vitamin K3 induces conformational changes in FtsZ that increase GTP hydrolysis and thereby, destabilize the FtsZ polymers. Together, our data provide evidence that Vitamin K3 derives its potent anti-pneumococcal activity by inhibiting FtsZ assembly.

RsmA3 modulates RpoS through the RetS-Gac-Rsm signalling pathway in response to H2 O2 stress in the phytopathogen Pseudomonas syringae.

Reactive oxygen species are a fatal challenge to the plant pathogenic bacterium Pseudomonas syringae. In this study, we reveal that the global regulatory protein RsmA3 from the RetS-Gac/Rsm signalling pathway modulates RpoS in the early-log growth phase in the P. syringae wild-type strain MB03, thereby regulating oxidative tolerance to H2 O2 and ultimately affecting pathogenicity to the host plant. Following increased H2 O2 by external addition or endogenous induction by menadione, the resistance of the mutant strain ΔretS to H2 O2 is significantly enhanced due to rapid increases in the transcription of Rsm-related non-coding small RNAs (nc sRNAs), a sigma factor RpoS, and H2 O2 -detoxifying enzymes. Moreover, the ΔretS mutant is significantly less pathogenic in cucumber leaves. Seven Rsm-related nc sRNAs (namely, rsmZ, rsmY and rsmX1-5 ) show functional redundancy in the RetS-Gac-Rsm signalling pathway. External addition of H2 O2 stimulates increases in the transcription of both rsmY and rsmZ. Thus, we propose a regulatory model of the RetS-Gac-Rsm signalling pathway in P. syringae MB03 for the regulation of H2 O2 tolerance and phytopathogenicity in the host plant.

Effects of dietary vitamin K3 supplementation on vitamin K1 and K2 (menaquinone) dynamics in dairy cows.

The effect of dietary vitamin K3 (VK3) on ruminant animals is not fully investigated. The aim of this study was to examine the effects of dietary VK3 on lactation performance, rumen characteristics, and VK1 and menaquinone (MK, or VK2) dynamics in the rumen, plasma, and milk of dairy cows. Eight Holstein dairy cows in late lactation periods were used in two crossover trials including a control (nontreatment) and a 50 or 200 mg/day (d) VK3 supplementation group. After 14 days, plasma, ruminal fluid, and milk were sampled and their VK1 and MKs contents were measured using fluorescence-high-performance liquid chromatography (HPLC). Milk production was unchanged after feeding 50 mg/day VK3 but marginally decreased after feeding 200 mg/day VK3. The molar ratio of propionate in ruminal fluid was significantly increased on feeding 200 mg/day VK3. Additionally, MK-4 concentrations significantly increased in both plasma and milk after VK3 feeding (50 and 200 mg/day). In ruminal fluid, MK-4 concentrations increased after 200 mg/day VK3 feeding. These results suggest that VK3 may be a good source of MK-4, the biologically active form of VK, in Holstein dairy cows during their late lactation periods. This study provides a basis for understanding the physiological role of VK in dairy cows.

A functional menadione biosynthesis pathway is required for capsule production by Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen that utilises a wide array of pathogenic and immune evasion strategies to cause disease. One immune evasion strategy, common to many bacterial pathogens, is the ability of S. aureus to produce a capsule that protects the bacteria from several aspects of the human immune system. To identify novel regulators of capsule production by S. aureus, we applied a genome wide association study (GWAS) to a collection of 300 bacteraemia isolates that represent the two major MRSA clones in UK and Irish hospitals: CC22 and CC30. One of the loci associated with capsule production, the menD gene, encodes an enzyme critical to the biosynthesis of menadione. Mutations in this gene that result in menadione auxotrophy induce the slow growing small-colony variant (SCV) form of S. aureus often associated with chronic infections due to their increased resistance to antibiotics and ability to survive inside phagocytes. Utilising such an SCV, we functionally verified this association between menD and capsule production. Although the clinical isolates with polymorphisms in the menD gene in our collections had no apparent growth defects, they were more resistant to gentamicin when compared to those with the wild-type menD gene. Our work suggests that menadione is involved in the production of the S. aureus capsule, and that amongst clinical isolates polymorphisms exist in the menD gene that confer the characteristic increased gentamicin resistance, but not the major growth defect associated with SCV phenotype.

Dietary vitamin K is remodeled by gut microbiota and influences community composition.

Vitamins have well-established roles in bacterial metabolism. Menaquinones (MKn, n = prenyl units in sidechain) are bacterially produced forms of vitamin K produced by the gut microbiota and consumed in the diet. Little is known about the influence of dietary vitamin K quinones on gut microbial composition and MKn production. Here, male and female C57BL6 mice were fed a vitamin K deficient diet or vitamin K sufficient diets containing phylloquinone (PK, plant-based vitamin K form), MK4, and/or MK9. DNA was extracted from cecal contents and 16S sequencing conducted to assess microbial composition. Cecal microbial community composition was significantly different in vitamin K deficient female mice compared to females on vitamin K sufficient diets (all p < .007). Parallel trends were seen in male mice, but were not statistically significant (all p > .05 but <0.1). Next, stable isotope-labeled vitamin K quinones were supplemented to male and female C57BL6 mice (2H7PK, 13C11MK4, 2H7MK7, 2H7MK9) and to an in vitro fermentation model inoculated with human stool (2H7PK, 2H7MK4, 2H7MK9, or vitamin K precursor 2H8-menadione). Vitamin K quinones in feces and culture aliquots were measured using LC-MS. In vivo, supplemented vitamin K quinones were remodeled to other MKn (2H7- or 13C6-labeled MK4, MK10, MK11, and MK12), but in vitro only the precursor 2H8-menadione was remodeled to 2H7MK4, 2H7MK9, 2H7MK10, and 2H7MK11. These results suggest that dietary vitamin K deficiency alters the gut microbial community composition. Further studies are needed to determine if menadione generated by host metabolism may serve as an intermediate in dietary vitamin K remodeling in vivo.

The Menadione-Mediated WST1 Reduction by Cultured Astrocytes Depends on NQO1 Activity and Cytosolic Glucose Metabolism.

The reduction of water-soluble tetrazolium salts (WSTs) is frequently used to determine the metabolic integrity and the viability of cultured cells. Recently, we have reported that the electron cycler menadione can efficiently connect intracellular oxidation reactions in cultured astrocytes with the extracellular reduction of WST1 and that this menadione cycling reaction involves an enzyme. The enzymatic reaction involved in the menadione-dependent WST1 reduction was found strongly enriched in the cytosolic fraction of cultured astrocytes and is able to efficiently use both NADH and NADPH as electron donors. In addition, the reaction was highly sensitive towards dicoumarol with Kic values in the low nanomolar range, suggesting that the NAD(P)H:quinone oxidoreductase 1 (NQO1) catalyzes the menadione-dependent WST1 reduction in astrocytes. Also, in intact astrocytes, dicoumarol inhibited the menadione-dependent WST1 reduction in a concentration-dependent manner with half-maximal inhibition observed at around 50 nM. Moreover, the menadione-dependent WST1 reduction by viable astrocytes was strongly affected by the availability of glucose. In the absence of glucose only residual WST1 reduction was observed, while a concentration-dependent increase in WST1 reduction was found during a 30 min incubation with maximal WST1 reduction already determined in the presence of 0.5 mM glucose. Mannose could fully replace glucose as substrate for astrocytic WST1 reduction, while other hexoses, lactate and the mitochondrial substrate ß-hydroxybutyrate failed to provide electrons for the cell-dependent WST1 reduction. These results demonstrate that the menadione-mediated WST1 reduction involves cytosolic NQO1 activity and that this process is strongly affected by the availability of glucose as metabolic substrate.

The effect of chronic dosing and p53 status on the genotoxicity of pro-oxidant chemicals in vitro.

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2',7'-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

[Selenoprotein thioredoxin reductase mediated menadione reduction: catalytic properties & inhibition effects].

Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec498 is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys498 is mainly responsible for menadione catalysis in comparison to Cys497, while the N-terminal Cys64 is slightly stronger than Cys59 regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.

Combining mutagenesis on Glu281 of prenyltransferase NovQ and metabolic engineering strategies for the increased prenylated activity towards menadione.

Prenyltransferase NovQ is a vital class involved in the biosynthesis of secondary metabolites such as clorobiocin and novobiocin. To investigate the relationship between structure and catalytic properties of NovQ, here, we have analyzed the substrate-binding site, namely PT barrel, and revealed that menadione hydroquinol formed intermolecular interactions with the residue Glu281 near the center of the active pocket. In this study, Glu281 was substituted with 9 diverse amino acids and catalytic properties of mutants were observed in vitro. Among them, E281Q showed 2.05-fold activities towards the aromatic substrate and prenyl donor, while others obtained catalytic efficiency between 8.4 and 88.6% of that of wild-type NovQ. Furthermore, the effects of catalytic conditions and substrate status on the activity of NovQ and its mutants were considered to obtain the optimized prenylated reaction. When the evolutionary NovQ variant E281Q was overexpressed in the host constructed to synthesize dimethylallyl diphosphate through the engineered mevalonate (MVA) pathway, we harvested up to 4.7 mg/L prenylated menadione at C-3 position by exogenously supplying the aromatic substrate. The construction of the microbial platform based on NovQ opens a new orientation to further biosynthesize various vitamin K2 with other ABBA prenyltransferases in E. coli.

Selenoprotein thioredoxin reductase mediated menadione reduction: catalytic properties & inhibition effects / 生物工程学报

Thioredoxin reductase (TrxR) is one class of the most important antioxidant selenoproteins and is involved in regulating tumor genesis and progression. It has been reported that naphthoquinones can target and inhibit TrxR1 activity therefore produce reactive oxygen species (ROS) mediated by TrxR1, resulting into cellular redox imbalance and making the naphthoquinone compounds to become potential antitumor chemotherapy drugs. The purpose of this work is to explore the interaction between TrxR1 and menadione using biochemical and mass-spectrometric (MS) analyses, to further reveal the detailed mechanisms of TrxR1-mediated naphthoquinone reduction and inhibition of TrxR1 by naphthoquinone compounds. Using the site-directed mutagenesis and recombinantly expressed TrxR1 variants, we measured the steady-state kinetic parameters of menadione reduction mediated by TrxR1 and its variants, performed the inhibition analysis of menadione on TrxR1 activity, and eventually identified the interaction between menadione and TrxR1 through MS analysis. We found that Sec-to-Cys mutation at residue of 498 significantly enhanced the efficiency of TrxR1-mediated menadione reduction, though the Sec⁴⁹⁸ is capable to catalyze the menadione reduction, indicating that TrxR1-mediated menadione reduction is dominantly in a Se-independent manner. Mutation experiments showed that Cys⁴⁹⁸ is mainly responsible for menadione catalysis in comparison to Cys⁴⁹⁷, while the N-terminal Cys⁶⁴ is slightly stronger than Cys⁵⁹ regarding the menadione reduction. LC-MS results detected that TrxR1 was arylated with one molecule of menadione, suggesting that menadione irreversibly modified the hyper-reactive Sec residue at the C-terminus of selenoprotein TrxR1. This study revealed that TrxR1 catalyzes the reduction of menadione in a Se-independent manner meanwhile its activity is irreversibly inhibited by menadione. Hereby it will be useful for the research and development of naphthoquinone anticancer drugs targeting TrxR1.

Revealing the Membrane-Bound Catalytic Oxidation of NADH by the Drug Target Type-II NADH Dehydrogenase.

Type-II NADH:quinone oxidoreductases (NDH-2s) are an important element of microbial pathogen electron transport chains and an attractive drug target. Despite being widely studied, its mechanism and catalysis are still poorly understood in a hydrophobic membrane environment. A recent report for the Escherichia coli NDH-2 showed NADH oxidation in a solution-based assay but apparently showed the reverse reaction in electrochemical studies, calling into question the validity of the electrochemical approach. Here we report electrochemical catalysis in the well-studied NDH-2 from Caldalkalibacillus thermarum (CthNDH-2). In agreement with previous reports, we demonstrated CthNDH-2 NADH oxidation in a solution assay and electrochemical assays revealed a system artifact in the absence of quinone that was absent in a membrane system. However, in the presence of either immobilized quinone or mobile quinone in a membrane, NADH oxidation was observed as in solution-phase assays. This conclusively establishes surface-based electrochemistry as a viable approach for interrogating electron transfer chain drug targets.

Nitroreductases of bacterial origin in Giardia lamblia: Potential role in detoxification of xenobiotics.

The anaerobic parasite Giardia lamblia, causative agent of persistent diarrhea, contains a family of nitroreductase genes most likely acquired by lateral transfer from anaerobic bacteria or archaebacteria. Two of these nitroreductases, containing a ferredoxin domain at their N-terminus, NR1, and NR2, have been characterized previously. Here, we present the characterization of a third member of this family, NR3. In functional assays, recombinant NR1 and NR3 reduced quinones like menadione and the antibiotic tetracycline, and-to much lesser extents-the nitro compound dinitrotoluene. Conversely, recombinant NR2 had no activity on tetracycline. Escherichia coli expressing NR3 were less susceptible to tetracycline, but more susceptible to the nitro compound metronidazole under semi-aerobic growth conditions. G. lamblia overexpressing NR1 and NR3, but not lines overexpressing NR2, are more susceptible to the nitro drug nitazoxanide. These findings suggest that NR3 is an active quinone reductase with a mode of action similar to NR1, but different from NR2. The biological function of this family of enzymes may reside in the use of xenobiotics as final electron acceptors. Thereby, these enzymes may provide at least two evolutionary advantages namely a higher potential to recycle NAD(P) as electron acceptors for the (fermentative) energy and intermediary metabolism, and the possibility to inactivate toxic xenobiotics produced by microorganisms living in concurrence inside the intestinal habitat.