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.

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.

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.

A Novel Biomimetic Tool for Assessing Vitamin K Status Based on Molecularly Imprinted Polymers.

Vitamin K was originally discovered as a cofactor required to activate clotting factors and has recently been shown to play a key role in the regulation of soft tissue calcification. This property of vitamin K has led to an increased interest in novel methods for accurate vitamin K detection. Molecularly Imprinted Polymers (MIPs) could offer a solution, as they have been used as synthetic receptors in a large variety of biomimetic sensors for the detection of similar molecules over the past few decades, because of their robust nature and remarkable selectivity. In this article, the authors introduce a novel imprinting approach to create a MIP that is able to selectively rebind vitamin K1. As the native structure of the vitamin does not allow for imprinting, an alternative imprinting strategy was developed, using the synthetic compound menadione (vitamin K3) as a template. Target rebinding was analyzed by means of UV-visible (UV-VIS) spectroscopy and two custom-made thermal readout techniques. This analysis reveals that the MIP-based sensor reacts to an increasing concentration of both menadione and vitamin K1. The Limit of Detection (LoD) for both compounds was established at 700 nM for the Heat Transfer Method (HTM), while the optimized readout approach, Thermal Wave Transport Analysis (TWTA), displayed an increased sensitivity with a LoD of 200 nM. The sensor seems to react to a lesser extent to Vitamin E, the analogue under study. To further demonstrate its potential application in biochemical research, the sensor was used to measure the absorption of vitamin K in blood serum after taking vitamin K supplements. By employing a gradual enrichment strategy, the sensor was able to detect the difference between baseline and peak absorption samples and was able to quantify the vitamin K concentration in good agreement with a validation experiment using High-Performance Liquid Chromatography (HPLC). In this way, the authors provide a first proof of principle for a low-cost, straightforward, and label-free vitamin K sensor.

Characterization of the Canine Anthracycline-Metabolizing Enzyme Carbonyl Reductase 1 (cbr1) and the Functional Isoform cbr1 V218.

The anthracyclines doxorubicin and daunorubicin are used in the treatment of various human and canine cancers, but anthracycline-related cardiotoxicity limits their clinical utility. The formation of anthracycline C-13 alcohol metabolites (e.g., doxorubicinol and daunorubicinol) contributes to the development of anthracycline-related cardiotoxicity. The enzymes responsible for the synthesis of anthracycline C-13 alcohol metabolites in canines remain to be elucidated. We hypothesized that canine carbonyl reductase 1 (cbr1), the homolog of the prominent anthracycline reductase human CBR1, would have anthracycline reductase activity. Recombinant canine cbr1 (molecular weight: 32.8 kDa) was purified from Escherichia coli. The enzyme kinetics of "wild-type" canine cbr1 (cbr1 D218) and a variant isoform (cbr1 V218) were characterized with the substrates daunorubicin and menadione, as well as the flavonoid inhibitor rutin. Canine cbr1 catalyzes the reduction of daunorubicin to daunorubicinol, with cbr1 D218 and cbr1 V218 displaying different kinetic parameters (cbr1 D218 Km: 188 ± 144 µM versus cbr1 V218 Km: 527 ± 136 µM, P < 0.05, and cbr1 D218 Vmax: 6446 ± 3615 nmol/min per milligram versus cbr1 V218 Vmax: 15539 ± 2623 nmol/min per milligram, P < 0.01). Canine cbr1 also metabolized menadione (cbr1 D218 Km: 104 ± 50 µM, Vmax: 2034 ± 307 nmol/min per milligram). Rutin acted as a competitive inhibitor for the reduction of daunorubicin (cbr1 D218 Ki: 1.84 ± 1.02 µM, cbr1 V218 Ki: 1.38 ± 0.47 µM). These studies show that canine cbr1 metabolizes daunorubicin and provide the necessary foundation to characterize the role of cbr1 in the variable pharmacodynamics of anthracyclines in canine cancer patients.

Pro-oxidant treatment of human prostate carcinoma (DU145) induces autoschizis cell death: autophagosomes build up out of injured endomembranes and mitochondria.

One hour after pro-oxidative treatment by either ascorbate (VC), menadione (VK3), or VC: VK3 combination followed by 24-h incubation in culture medium, DU145 human prostate carcinoma cells developed ultrastructural-dependent organelle damage with the sequence Sham > VC > VK3 > VC: VK3. Along the nuclear alterations and the cytoplasm self-excisions reducing cell size, other induced injuries concerned mitochondria and endomembranes that associated with lysosomes. Damaged organelles surrounded by specialized endoplasmic membranes formed autophagosomes out of phagophores that also captured pieces of glycogen-rich cytoplasm. Most autophagosomes amassed in the diminished-size perikarya and corroborated the enhanced cytotoxicity of the VC: VK3 treatment. These accumulations did not initiate cell death, instead were merely signs of excessive "recycling" of damaged organelles. These features may reflect that high lysosomal activities provided foodstuffs in an ultimate strategy of survival of the tumor cells already devastated by reactive oxidative species (ROS) energetic sites. As such they became transient markers preceding cell death induced to occur by autoschizis and not by apoptosis or other cell deaths. This report could provide more support for the usage of this vitamin combination named APATONE as inexpensive potent adjuvant or treatment in prostate cancers.

Intracellular forms of menadione-dependent small-colony variants of methicillin-resistant Staphylococcus aureus are hypersusceptible to ß-lactams in a THP-1 cell model due to cooperation between vacuolar acidic pH and oxidant species.

OBJECTIVES: Phagocytosed methicillin-resistant Staphylococcus aureus (MRSA) are susceptible to ß-lactams because of an acid-induced conformational change of penicillin-binding protein (PBP) 2a within phagolysosomes. We have examined whether this mechanism applies to menD and hemB small-colony variants (SCVs) of the COL MRSA strain, using cloxacillin, meropenem, doripenem, and vancomycin as comparator. METHODS: Intracellularly, the change in cfu from post-phagocytosis inoculum was measured after 24 h of incubation with antibiotics combined or not with N-acetylcysteine (NAC; oxidant species scavenger); the relative potency (C(s)) was calculated from the Hill equation of concentration-response curves. Extracellularly, the effect of a pre-incubation with H(2)O(2) was determined on MICs and killing at pH 7.4 and 5.5. RESULTS: Intracellularly, the ß-lactam C(s) was similar for the COL strain and the hemB mutant and not modified or slightly decreased (2- to 16-fold) by NAC. In contrast, the C(s) was 100- to 900-fold lower for the menD mutant, but similar to that for the COL strain when NAC was present. Extracellularly, ß-lactam MICs were markedly reduced at pH 5.5 for the parental strain and the haemin-supplemented hemB mutant, with limited additional effect of pre-incubation with H(2)O(2). In contrast, MICs remained elevated at pH 5.5 for the menD mutant (supplemented with menadione sodium bisulphite or not), but were 7-10 dilutions lower after pre-incubation with H(2)O(2). Vancomycin MICs were unaltered in all conditions, with no marked effect of NAC on C(s). CONCLUSIONS: Cooperation between acidic pH and oxidant species confers high potency to ß-lactams against intracellular forms of menD SCVs of MRSA.

Vitamin E decreases extra-hepatic menaquinone-4 concentrations in rats fed menadione or phylloquinone.

SCOPE: The mechanism for increased bleeding and decreased vitamin K status accompanying vitamin E supplementation is unknown. We hypothesized that elevated hepatic α-tocopherol (α-T) concentrations may stimulate vitamin K metabolism and excretion. Furthermore, α-T may interfere with the side chain removal of phylloquinone (PK) to form menadione (MN) as an intermediate for synthesis of tissue-specific menaquinone-4 (MK-4). METHODS AND RESULTS: In order to investigate these hypotheses, rats were fed phylloquinone (PK) or menadione (MN) containing diets (2 µmol/kg) for 2.5 weeks. From day 10, rats were given daily subcutaneous injections of either α-T (100 mg/kg) or vehicle and were sacrificed 24 h after the seventh injection. Irrespective of diet, α-T injections decreased MK-4 concentrations in brain, lung, kidney, and heart; and PK in lung. These decreases were not accompanied by increased excretion of urinary 5C- or 7C-aglycone vitamin K metabolites, however, the urinary α-T metabolite (α-CEHC) increased ≥ 100-fold. Moreover, α-T increases were accompanied by downregulation of hepatic cytochrome P450 expression and modified expression of tissue ATP-binding cassette transporters. CONCLUSION: Thus, in rats, high tissue α-T depleted tissue MK-4 without significantly increasing urinary vitamin K metabolite excretion. Changes in tissue MK-4 and PK levels may be a result of altered regulation of transporters.

Pharmacodynamic evaluation of the activity of antibiotics against hemin- and menadione-dependent small-colony variants of Staphylococcus aureus in models of extracellular (broth) and intracellular (THP-1 monocytes) infections.

Staphylococcus aureus small-colony variants (SCVs) persist intracellularly, which may contribute to persistence/recurrence of infections and antibiotic failure. We have studied the intracellular fate of menD and hemB mutants (corresponding to menadione- and hemin-dependent SCVs, respectively) of the COL methicillin-resistant S. aureus (MRSA) strain and the antibiotic pharmacodynamic profile against extracellular (broth) and intracellular (human THP-1 monocytes) bacteria. Compared to the parental strain, SCVs showed slower extracellular growth (restored upon medium supplementation with menadione or hemin), reduced phagocytosis, and, for the menD SCV, lower intracellular counts at 24 h postinfection. Against extracellular bacteria, daptomycin, gentamicin, rifampin, moxifloxacin, and oritavancin showed similar profiles of activity against all strains, with a static effect obtained at concentrations close to their MICs and complete eradication as maximal effect. In contrast, vancomycin was not bactericidal against SCVs. Against intracellular bacteria, concentration-effect curves fitted sigmoidal regressions for vancomycin, daptomycin, gentamicin, and rifampin (with maximal effects lower than a 2-log decrease in CFU) but biphasic regressions (with a maximal effect greater than a 3-log decrease in CFU) for moxifloxacin and oritavancin, suggesting a dual mode of action against intracellular bacteria. For all antibiotics, these curves were indistinguishable between the strains investigated, except for the menD mutant, which systematically showed a lower amplitude of the concentration-effect response, with markedly reduced minimal efficacy (due to slower growth) but no change in maximal efficacy. The data therefore show that the maximal efficacies of antibiotics are similar against normal-phenotype and menadione- and hemin-dependent strains despite their different intracellular fates, with oritavancin, and to some extent moxifloxacin, being the most effective.

Delay of gap filling during nucleotide excision repair by base excision repair: the concept of competition exemplified by the effect of propolis.

Nucleotide excision repair (NER) consists of a sequence of events including DNA damage recognition, excision of the damage containing oligonucleotide, gap filling, and ligation. We found that gap filling during the repair of ultraviolet (UV)C-induced DNA lesions was inhibited by various compounds, e.g., amoxicillin, and mixtures, e.g., propolis, the materials that could induce oxidative DNA damage in serum-supplemented cell cultures. Such inhibitory effect was also demonstrated by the immunostaining experiment and host cell reactivation assay. In this study, we link the repair of oxidative DNA damage with the inhibition of gap filling. Our experimental evidence includes the following: (1) induction of oxidative DNA damage and inhibition of gap filling were quantitatively correlated; (2) although the repair of UV-induced DNA damage was delayed in the presence of propolis, the repair of propolis-induced oxidative DNA damage proceeded regardless of preexposure to UV radiation; (3) inhibition of gap filling by propolis was absent in base excision repair (BER)-deficient cells; (4) suppression of propolis-induced oxidative DNA damage by ß-carotene abolished the inhibition of gap filling; and (5) inhibition of gap filling was also found with typical BER-inducing agents such as hydrogen peroxide, menadione, and methyl methanesulfonate. We propose that competition may occur between NER and BER, which results in delay of gap filling. Our study reveals the dominancy of BER over NER.

Plasma vitamin K concentration in horses supplemented with several vitamin K homologs.

The effect of several vitamin K homologs on plasma vitamin K concentration was determined to assess their potential as a vitamin K supplement for adult horses. Sixteen Thoroughbred horses consisting of 8 mares and 8 geldings, aged 8.4 ± 3.6 yr and weighing 520.8 ± 36.1 kg, were allocated to 4 groups (n = 4). Each group was given phylloquinone, menaquinone-4, or menadione at 58 µmol/d, or no vitamin K supplement for 7 d. Plasma samples were collected before feeding, and 2, 4, and 8 h after feeding on d 7, and plasma concentrations of phylloquinone and menaquinone-4 were determined. Plasma phylloquinone concentration was greater in the phylloquinone group than in the other groups (P < 0.001). The phylloquinone concentration quadratically increased (P < 0.001) after feeding in the phylloquinone group but no changes in the plasma phylloquinone concentration were observed after feeding in the other groups. Plasma menaquinone-4 concentration was greater (P < 0.001) in the menadione group than the other groups, including the menaquinone-4 group. Menaquinone-4 concentration did not change (P = 0.192) after feeding in each group. Menaquinone-4 has been considered the most potent vitamin K homolog for bone metabolism; therefore, the present experiment indicates that menadione is a good source of vitamin K for bone health in horses because it is the only vitamin K homolog that increased the plasma concentrations of menaquinone-4.

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents.

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 µM of XTT and 60 µM of menadione in 250 µl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were >0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli.

Response to different oxidants of Saccharomyces cerevisiae ure2Delta mutant.

Growth of Saccharomyces cerevisiae ure2Delta mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H(2)O(2) was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Delta mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the gamma-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level.

Induction of antioxidant and phase 2 drug-metabolizing enzymes by falcarindiol isolated from Notopterygium incisum extract, which activates the Nrf2/ARE pathway, leads to cytoprotection against oxidative and electrophilic stress.

In the present study, we isolated falcarindiol from Notopterygium incisum and investigated the effect of falcarindiol on the expression of antioxidant enzymes (AOEs), such as catalase, and phase 2 drug-metabolizing enzymes (DMEs), such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1, in a cultured cell line from normal rat liver, Clone 9 cells. Exposure of Clone 9 cells to falcarindiol resulted in the significant induction of AOEs and phase 2 DMEs. Western blot analysis and transfection studies using a luciferase reporter construct demonstrated that the induction of AOEs and phase 2 DMEs by falcarindiol was caused through the Nrf2/ARE (nuclear factor-E2-related factor 2/antioxidant response element) pathway. Pretreatment of cells with falcarindiol accelerated the detoxification of a potentially toxic quinone (menadione) and mitigated menadione-induced cytotoxicity. We found that falcarindiol was a novel inducer of AOEs and phase 2 DMEs and falcarindiol might exhibit chemopreventive activity.

NRH:quinone oxidoreductase 2 (NQO2) catalyzes metabolic activation of quinones and anti-tumor drugs.

NRH:quinone oxidoreductase 2 (NQO2) is a cytosolic flavoprotein that utilizes NRH as electron donor. The present studies investigate the role of NQO2 in metabolic detoxification/activation of quinones and quinone based anti-tumor drugs. Chinese hamster ovary (CHO) cells stably overexpressing cDNA derived mouse NQO2 and mouse keratinocytes from DMBA-induced skin tumors in wild-type and NQO2-null mice were generated. The CHO cells overexpressing NQO2 and mouse keratinocytes expressing or deficient in NQO2 were treated with varying concentrations of mitomycin C (MMC), CB1954, MMC analog BMY25067, EO9, menadione and BP-3,6-quinone, in the absence and presence of NRH. The cytotoxicity of the drugs was evaluated by colony formation. The CHO cells overexpressing higher levels of mouse NQO2 showed significantly increased cytotoxicity to menadione, BP-3,6-quinone and to the anti-tumor drugs MMC and CB1954 when compared to CHO cells expressing endogenous NQO2. The cytotoxicity increased in presence of NRH. Similar results were also observed with BMY25067 and EO9 treatments, but to a lesser extent. The results on keratinocytes deficient in NQO2 supported the data from CHO cells. The inclusion of NRH had no effect on cytotoxicity of quinones and drugs in keratinocytes deficient in NQO2. Mouse NQO2 protein was expressed in bacteria, purified and used to study the role of NQO2 in MMC-induced DNA cross-linking. Bacterially expressed and purified NQO2 efficiently catalyzed MMC activation that led to DNA cross-linking. These results concluded that NQO2 plays a significant role in the metabolic activation of both quinones and anti-tumor drugs leading to cytotoxicity and cell death.

Recruitment of a foreign quinone into the A1 site of photosystem I. In vivo replacement of plastoquinone-9 by media-supplemented naphthoquinones in phylloquinone biosynthetic pathway mutants of Synechocystis sp. PCC 6803.

Interruption of the phylloquinone (PhQ) biosynthetic pathway by interposon mutagenesis of the menA and menB genes in Synechocystis sp. PCC 6803 results in plastoquinone-9 (PQ-9) occupying the A(1) site and functioning in electron transfer from A(0) to the FeS clusters in photosystem (PS) I (Johnson, T. W., Shen, G., Zybailov, B., Kolling, D., Reategui, R., Beauparlant, S., Vassiliev, I. R., Bryant, D. A., Jones, A. D., Golbeck, J. H., and Chitnis, P. R. (2000) J. Biol. Chem. 275, 8523-8530. We report here the isolation of menB26, a strain of the menB mutant that grows in high light by virtue of a higher PS I to PS II ratio. PhQ can be reincorporated into the A(1) site of the menB26 mutant strain by supplementing the growth medium with authentic PhQ. The reincorporation of PhQ also occurs in cells that have been treated with protein synthesis inhibitors, consistent with a displacement of PQ-9 from the A(1) site by mass action. The doubling time of the menB26 mutant cells, but not the menA mutant cells, approaches the wild type when the growth medium is supplemented with naphthoquinone (NQ) derivatives such as 2-CO(2)H-1,4-NQ and 2-CH(3)-1,4-NQ. Since PhQ replaces PQ-9 in the supplemented menB26 mutant cells, but not in the menA mutant cells, the phytyl tail accompanies the incorporation of these quinones into the A(1) site. Studies with menB26 mutant cells and perdeuterated 2-CH(3)-1,4-NQ shows that phytylation occurs at position 3 of the NQ ring because the deuterated 2-methyl group remains intact. Therefore, the specificity of the phytyltransferase enzyme is selective with respect to the group present at ring positions 2 and 3. Supplementing the growth medium of menB26 mutant cells with 1,4-NQ also leads to its incorporation into the A(1) site, but typically without either the phytyl tail or the methyl group. These findings open the possibility of biologically incorporating novel quinones into the A(1) site by supplementing the growth medium of menB26 mutant cells.