Simple menaquinones reduce carbon tetrachloride and iron (III)

Cell-free supernatant from Shewanella oneidensis MR-1 reduced carbon tetrachloride to chloroform, a suspension of Fe(III) and solid Fe(III) to iron (II). The putative reducing agent was tentatively identified as menaquinone-1 (MQ-1)—a water-soluble menaquinone with a single isoprenoid residue in the side chain. Synthetic MQ-1 reduced carbon tetrachloride to chloroform and amorphous iron (III) hydroxide to iron (II). To test the generality of this result among menaquinones, the reductive activities of vitamin K₂ (MQ-7)—a lipid-associated menaquinone with 7 or 8 isoprenoid residues—was evaluated. This molecule also reduced carbon tetrachloride to chloroform and iron (III) to iron (II). The results indicate that molecules within the menaquinone family may contribute to both the extracellular and cell-associated reduction of carbon tetrachloride and iron (III).     

Electron transport driven phosphorylation catalyzed by proteoliposomes containing hydrogenase,fumarate reductase and ATP synthase

Liposomes containing the hydrogenase complex and the fumarate reductase complex isolated from Wolinella (formerly Vibrio) succinogenes, together with vitamin K₁ catalyzed the electron transport from H₂ to fumarate. With the fumarate reductase complex present in excess the activity of electron transport was close to that of the hydrogenase complex. Liposomes containing an ATP synthase in addition to the electron transport components catalyzed the phosphorylation of ADP driven by the electron transport.     

Effects of flavonoids on membrane adaptation of food-associated bacteria

The effects of naringenin and the biflavonoids amentoflavone and tetrahydroamentoflavone on select bacterial lipids (carotenoids, fatty acids, and menaquinones) and membrane fluidity based on Laurdan generalized polarization were investigated. For this purpose, the pigment-forming food-associated microorganisms Staphylococcus xylosus (DSM 20266^T and J70), Staphylococcus carnosus DSM 20501^T, and Micrococcus luteus (ATCC 9341 and J3) were studied.The results suggest an envelope stress response by microorganisms due to flavonoids and an employment of adaptive mechanisms using carotenoids, fatty acids, and menaquinones. The flavonoid monomer naringenin impacted carotenoids, fatty acids, menaquinones, and membrane fluidity. Naringenin significantly influenced the carotenoid profile, particularly by an increase in the relative proportion of 4,4′-diaponeurosporenoic acid in Staphylococcus xylosus. Amentoflavone caused changes mainly in the membrane of Micrococcus luteus and decreased the menaquinone content. Tetrahydroamentoflavone mainly affected the carotenoids in the investigated strains.The noticeably different CCS value of tetrahydroamentoflavone compared to naringenin and amentoflavone revealed further insights into the structure-dependent effects of flavonoids.This study provides valuable insights into the response of pigment-forming food-associated microorganisms to naringenin, amentoflavone, and tetrahydroamentoflavone, which is important for the targeted and safe application of the latter as natural preservatives and useful for further research on the mechanisms of action.     

Succinate:quinone oxidoreductases from ϵ-proteobacteria

The ϵ-proteobacteria form a subdivision of the Proteobacteria including the genera Wolinella, Campylobacter, Helicobacter, Sulfurospirillum, Arcobacter and Dehalospirillum. The majority of these bacteria are oxidase-positive microaerophiles indicating an electron transport chain with molecular oxygen as terminal electron acceptor. However, numerous members of the ϵ-proteobacteria also grow in the absence of oxygen. The common presence of menaquinone and fumarate reduction activity suggests anaerobic fumarate respiration which was demonstrated for the rumen bacterium Wolinella succinogenes as well as for Sulfurospirillum deleyianum, Campylobacter fetus, Campylobacter rectus and Dehalospirillum multivorans. To date, complete genome sequences of Helicobacter pylori and Campylobacter jejuni are available. These bacteria and W. succinogenes contain the genes frdC, A and B encoding highly similar heterotrimeric enzyme complexes belonging to the family of succinate:quinone oxidoreductases. The crystal structure of the W. succinogenes quinol:fumarate reductase complex (FrdCAB) was solved recently, thus providing a model of succinate:quinone oxidoreductases from ϵ-proteobacteria. Succinate:quinone oxidoreductases are being discussed as possible therapeutic targets in the treatment of several pathogenic ϵ-proteobacteria.     

Low-potential cytochrome b as an essential electron-transport component of menaquinone reduction by formate in Vibrio succinogenes

Incorporation of the electron-transport enzymes of Vibrio succinogenes into liposomes was used to investigate the question of whether, in this organism, a cytochrome b is involved in electron transport from formate to fumarate on the formate side of menaquinone. (1) Formate dehydrogenase lacking cytochrome b was prepared by splitting the cytochrome from the formate dehydrogenase complex. The enzyme consisted of two different subunits (M_r 110 000 and 20 000), catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone by formate, and could be incorporated into liposomes. (2) The modified enzyme did not restore electron transport from formate to fumarate when incorporated into liposomes together with vitamin K-1 (instead of menaquinone) and fumarate reductase complex. In contrast, restoration was observed in liposomes that contained formate dehydrogenase with cytochrome b (E_m = ?224 mV), in addition to the subunits mentioned above (formate dehydrogenase complex). (3) In the liposomes containing formate dehydrogenase complex and fumarate reductase complex, the response of the cytochrome b of the formate dehydrogenase complex was consistent with its interaction on the formate side of menaquinone in a linear sequence of the components. The low-potential cytochrome b associated with fumarate reductase complex was not reducible by formate under any condition. It is concluded that the low-potential cytochrome b of the formate dehydrogenase complex is an essential component in the electron transport from formate to menaquinone. The low-potential cytochrome b of the fumarate reductase complex could not replace the former cytochrome in restoring electron-transport activity.     

Biotransformation of menadione to its prenylated derivative MK-3 using recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Prenylated quinones, especially menaquinones, have significant physiological activities, but are arduous to synthesize efficiently. Due to the relaxed aromatic substrate specificity and prenylation regiospecificity at the ortho- site of the phenolic hydroxyl group, the aromatic prenyltransferase NovQ from Streptomyces may be useful in menaquinone synthesis from menadione. In this study, NovQ was overexpressed in Pichia pastoris. After fermentation optimization, NovQ production increased by 1617%. Then the different effects of metal ions, detergents and pH on the activity of purified NovQ were investigated to optimize the prenylation reaction. Finally, purified NovQ and cells containing NovQ were used for menadione prenylation in vitro and in vivo, respectively. Menaquinone-1 (MK-1) was detected as the only product in vitro with γ,γ-dimethylallyl pyrophosphate and menadione hydroquinol substrates. MK-3 at a concentration of 90.53 mg/L was detected as the major product of whole cell catalysis with 3-methyl-2-buten-1-ol and menadione hydroquinol substrates. This study realized whole cell catalysis converting menadione to menaquinones.     

Isoprenoid quinone, cellular fatty acid composition and diaminopimelic acid isomers of newly classified thermophilic anaerobic Gram-positive bacteria

The configuration of quinone systems, cellular fatty acids and diaminopimelic acids in 11 species of thermophilic clostridia which have been classified into new genera based on their 16S rRNA sequences was determined. It was found that menaquinone 7 was present in 10 species as the major component of menaquinone systems by analyses using HPLC with photodiode-array detector and electron impact mass spectrometry. In seven of the 11 species, the major cellular fatty acids were determined to be iso-15:0 and iso-17:0. As a results of chemotaxonomic studies it was demonstrated that the representatives of the new genera Thermoanaerobacter and Thermoanaerobacterium were heterogeneous in their chemical composition, whereas strains of the new genus Moorella and of the new unnamed genus which is composed of (Clostridium) stercorarium and (Clostridium) thermolacticum showed homogeneity in their chemotaxonomic characteristics.     

Isolation and properties of naphthoate synthetase from Mycobacterium phlei

Cell-free extracts obtained by sonication of Mycobacterium phlei cells contain an important enzyme of the menaquinone (= vitamin K₂) biosynthetic pathway. This enzyme, naphthoate synthetase (1,4-dihydroxy-2-naphthoate synthetase), was partially purified by chromatography on Sepharose 6BCL. Conversion of o-succinylbenzoate to 1,4-dihydroxy-2-naphthoate was followed by a radioactivity assay using o-[2,3-¹⁴C₂]succinylbenzoate, or by a spectrophotofluorometric assay. o-[1-¹³C]Succinylbenzoate was converted intact by the extracts to dihydroxynaphthoate containing ¹³C only in the carboxyl carbon atom. For maximum activity, the enzyme requires ATP, Mg²⁺, and coenzyme A. The pH optimum is 6.9 and the molecular weight approximately 44,000. In the presence of farnesyl pyrophosphate, the extracts convert o-[2,3-¹⁴C₂]succinylbenzoate to ¹⁴C-containing menaquinone.     

Simultaneous formation of menaquinones and demethylmenaquinones byMicrococcus varians IAM 12146 depending on cell growth media

Changes in the isoprenoid quinone composition ofMicrococcus varians IAM 12146 in response to growth in different media were investigated. When the bacterium was growth in an ordinary complex medium, it produced menaquinones as the sole quinones, with a dihydrogenated menaquinone with seven isoprene units as the major component, at all growth stages. On the other hand, cells grown in a chemically defined medium containing glutamate and pyruvate as carbon sources produced both menaquinones and demethylmenaquinones. The major demethylmenaquinone homologs produced were the unsaturated and dihydrogenated types with seven isoprene units. The demethylmenaquinone/menaquinone ratio in cells varied during a batch growth in the chemically defined medium. The highest ratio was found in cells at the mid-exponential phase of growth.     

Role of Menaquinones in Fe(III) Reduction by Membrane Fractions of Shewanella putrefaciens

Two Tn5-generated mutants of Shewanella putrefaciens with insertions in menD and menB were isolated and analyzed. Both mutants were deficient in the use of several terminal electron acceptors, including Fe(III). This deficiency was overcome by the addition of menaquinone (vitamin K(2)). Isolated membrane fractions from both mutants were unable to reduce Fe(III) in the absence of added menaquinone when formate was used as the electron donor. These results indicate that menaquinones are essential components for the reduction of Fe(III) by both whole cells and purified membrane fractions when formate or lactate is used as the electron donor.     

Menaquinone is an obligatory component of the chain catalyzing succinate respiration in Bacillus subtilis

The question was investigated as to whether the bacterial menaquinone (MK) is a component of the electron transport chain catalyzing succinate respiration in Bacillus subtilis. Three different methods were applied, and the following consistent results were obtained. (i) Solvent extraction of MK from the bacterial membrane caused total inhibition of the respiratory activities with succinate and NADH, while the activity of succinate dehydrogenase remained unaffected. The respiratory activities were restored onincorporation of vitamin K₁ into the membrane preparation. (ii) The membrane fraction of a B. subtilis mutant containing 15% of the wild-type amount of MK, respired succinate and NADH at reduced activities. Wild-type activities were restored on fusion of the preparation to liposomes containing vitamin K₁. (iii) The membrane fraction of B. subtilis catalyzed succinate oxidation by various water-soluble naphtho- or benzoquinones at specific activities exceeding to that of succinate respiration. The results suggest that MK is involved in succinate respiration, although its redox potential is unfavorable.Abbreviations MK menaquinone - MKH₂ reduced menaquinone - E₀' standard redox potential at pH 7 - PMS phenazine methosulfate - DCPIP 2,6-Dichlorophenol-indophenol - Q ubiquinone - Q₀ 2,3-dimethoxy-5-methyl-1,4-bezoquinone - DMN, 2,3 dimethyl-1,4-naphthoquinone - DMK demethylmenaquinone     

Role of the entC gene in enterobactin and menaquinone biosynthesis in Escherichia coli

Tn10 mutants of Escherichia coli MC4100 were screened for their inability to grow under iron deficiency and for their inability to grow under anaerobiosis in the presence of fumarate as an electron acceptor. A strain so obtained (E. coli PBB1) lacked the ability to convert chorismic acid to isochorismic acid. This shows that the gene (entC) encoding isochorismate synthase was mutated. E. coli PBB1 did not produce any detectable amounts of menaquinones (vitamin K2) or enterobactin. When supplemented with isochorismic acid this strain produced menaquinones, indicating that isochorismic acid is involved not only in enterobactin but also in menaquinone biosynthesis. The entC gene was isolated and was shown to be part of the enterobactin gene cluster: It was located on a DNA fragment (9 kb in length) which also carried the entA gene. The DNA fragment was identified by restriction site mapping and was compared to a previously published map of the enterobactin gene cluster. The entC gene on this fragment responds not only to conditions (iron deficiency) that stimulate enterobactin biosynthesis but also to anaerobiosis which results in increased isochorismic acid formation and increased menaquinone biosynthesis. We conclude that isochorismic acid, isochorismic synthase, and the gene (entC) encoding this enzyme are involved in catalytic events at a metabolic branch point from which both enterobactin and menaquinones originate.     

Menaquinone composition of some micrococci determined by high performance liquid chromatography

The menaquinone composition of some species of the genus Micrococcus was analysed by high performance liquid chromatography. Both unsaturated and hydrogenated menaquinones were detected. Micrococcus kristinae, M. lylae and M. nishinomiyaensis contained hydrogenated menaquinones while M. sedentarius contained unsaturated menaquinone. The predominant menaquinone isoprenologues of these species are MK- 7 (H₂), MK- 8 (H₂), MK- 8 (H₂) and MK- 8 , respectively. These observations confirm further the heterogeneity of the genus.     

Measurement of vitamin K in human liver by gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection

A sensitive and precise method for measuring endogenous phylloquinone (K₁) and menaquinone (MK-n) in human liver was developed, based on gradient elution high-performance liquid chromatography using platinum-black catalyst reduction and fluorimetric detection. Subnanogram levels of vitamin K compounds in 1 g of liver specimen were detectable. We measured vitamin K concentrations in 38 human resected livers. K₁ and MK-4 to MK-13 were detected. The concentrations of MK-10 to MK-12 in livers with chronic hepatitis (n=10) and cirrhosis (n=22) were significantly lower than in normal livers (n=6). It is suggested that the decreased concentrations indicate functional damage of the hepatocytes.     

Shewanella litorisediminis sp. nov., a gammaproteobacterium isolated from a tidal flat sediment

A Gram-negative, facultatively anaerobic, non-motile and rod-shaped bacterial strain, designated SMK1-12^T, was isolated from a tidal flat sediment on the western coast of Korea. Phylogenetic analyses based on 16S rRNA and gyrB gene sequences showed that strain SMK1-12^T belonged to the genus Shewanella, clustering with the type strain of Shewanella amazonensis. Strain SMK1-12^T exhibited the highest 16S rRNA gene sequence similarity value (97.0 %) and the highest gyrB sequence similarity value (87.8 %) to S. amazonensis SB2B^T, respectively. Strain SMK1-12^T contained simultaneously both menaquinones and ubiquinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8. The major fatty acids (>10 % of the total fatty acids) detected in strain SMK1-12^T were the MIDI system summed feature 3 (iso-C_15:0 2-OH and/or C_16:1 ω7c), iso-C_15:0, C_17:1 ω8c and C_16:0. The DNA G+C content of strain SMK1-12^T was 58.0 mol% and its mean DNA–DNA relatedness value with S. amazonensis ATCC 700329^T was 15 ± 4.6 %. Differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain SMK1-12^T is distinguishable from recognized Shewanella species. On the basis of the data presented, strain SMK1-12^T is considered to represent a novel Shewanella species, for which the name Shewanella litorisediminis sp. nov. is proposed. The type strain is SMK1-12^T (=KCTC 23961^T = CCUG 62411^T).     

A note on the separation of natural mixtures of bacterial menaquinones using reverse-phase high-performance liquid chromatography

Natural mixtures of bacterial menaquinones were separated according to the length and degree of hydrogenation of the polyisoprenoid side-chain using reverse-phase high-performance liquid chromatography. The chromatographic system described affords a sensitive and rapid means of menaquinone characterization.     

The specific functions of menaquinone and demethylmenaquinone in anaerobic respiration with fumarate,dimethylsulfoxide, trimethylamine N-oxide and nitrate by Escherichia coli

The respiratory activities of E. coli with H₂ as donor and with nitrate, fumarate, dimethylsulfoxide (DMSO) or trimethylamine N-oxide (TMAO) as acceptor were measured using the membrane fraction of quinone deficient strains. The specific activities of the membrane fraction lacking naphthoquinones with fumarate, DMSO or TMAO amounted to 2% of those measured with the membrane fraction of the wild-type strain. After incorporation of vitamin K₁ [instead of menaquinone (MK)] into the membrane fraction deficient of naphthoquinones, the activities with fumarate or DMSO were 92% or 17%, respectively, of the activities which could be theoretically achieved. Incorporation of demethylmenaquinone (DMK) did not lead to a stimulation of the activities of the mutant. In contrast, the electron transport activity with TMAO was stimulated by the incorporation of either vitamin K₁ or DMK. Nitrate respiration was fully active in membrane fractions lacking either naphthoquinones or Q, but was 3% of the wild-type activity, when all quinones were missing. Nitrate respiration was stimulated on the incorporation of either vitamin K₁ or Q into the membrane fraction lacking quinones, while the incorporation of DMK was without effect. These results suggest that MK is specifically involved in the electron transport chains catalyzing the reduction of fumarate or DMSO, while either MK or DMK serve as mediators in TMAO reduction. Nitrate respiration requires either Q or MK.Abbreviations DMK demethylmenaquinone - MK menaquinone - Q ubiquinone - DMSO dimethylsulfoxide - TMAO trimethylamine N-oxide - DMS dimethylsulfide - TMA trimethylamine - BV benzylviologen     

Sequence analysis and specificity of distinct types of menaquinone methyltransferases indicate the widespread potential of methylmenaquinone production in bacteria and archaea

Menaquinone (MK) serves as an essential membranous redox mediator in various electron transport chains of aerobic and anaerobic respiration. In addition, the composition of the quinone/quinol pool has been widely used as a biomarker in microbial taxonomy. The HemN-like class C radical SAM methyltransferases (RSMTs) MqnK, MenK and MenK2 have recently been shown to facilitate specific menaquinone methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesize 8-methylmenaquinone, 7-methylmenaquinone and 7,8-dimethylmenaquinone. However, the vast majority of protein sequences from the MqnK/MenK/MenK2 family belong to organisms, whose capacity to produce methylated menaquinones has not been investigated biochemically. Here, representative putative menK and menK2 genes from Collinsella tanakaei and Ferrimonas marina were individually expressed in Escherichia coli (wild-type or ubiE deletion mutant) and the corresponding cells were found to produce methylated derivatives of the endogenous MK and 2-demethylmenaquinone. Cluster and phylogenetic analyses of 828 (methyl)menaquinone methyltransferase sequences revealed signature motifs that allowed to discriminate enzymes of the MqnK/MenK/MenK2 family from other radical SAM enzymes and to identify C-7-specific menaquinone methyltransferases of the MenK2 subfamily. This study will help to predict the methylation status of the quinone/quinol pool of a microbial species (or even a microbial community) from its (meta)genome and contribute to the future design of microbial quinone/quinol pools in a Synthetic Biology approach.     

Improving coenzyme Q8 production in Escherichia coli employing multiple strategies

Coenzyme Q (CoQ) is a medically valuable compound and a high yielding strain for CoQ will have several benefits for the industrial production of CoQ. To increase the CoQ₈ content of E. coli, we blocked the pathway for the synthesis of menaquinone by deleting the menA gene. The blocking of menaquinone pathway increased the CoQ₈ content by 81 % in E. coli (ΔmenA). To study the CoQ producing potential of E. coli, we employed previous known increasing strategies for systematic metabolic engineering. These include the supplementation with substrate precursors and the co-expression of rate-limiting genes. The co-expression of dxs-ubiA and the supplementation with substrate precursors such as pyruvate (PYR) and parahydroxybenzoic acid (pHBA) increased the content of CoQ₈ in E. coli (ΔmenA) by 125 and 59 %, respectively. Moreover, a 180 % increase in the CoQ₈ content in E. coli (ΔmenA) was realized by the combination of the co-expression of dxs-ubiA and the supplementation with PYR and pHBA. All in all, CoQ₈ content in E. coli increased 4.06 times by blocking the menaquinone pathway, dxs-ubiA co-expression and the addition of sodium pyruvate and parahydroxybenzoic acid to the medium. Results suggested a synergistic effect among different metabolic engineering strategies.