Quinones from Archaebacteria, I. New types of menaquinones from the thermophilic archaebacterium Thermoproteus tenax

From the archaebacterium Thermoproteus tenax, strain Kra-1 a mixture of 6 quinones of menaquinone and phylloquinone type with isopentyl side chains, MK-6(12H), MK-6-(10H), MK-5(10H), MK-5(8H), MK-4(8H), MK-4(6H) and two analogous quinones, containing in addition a methyl group in the naphthoquinone system, were isolated and characterized.     

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.     

Fully saturated menaqionones in the archaebacterium Pyrobaculum islandicum

Abstract The anaerobic, thermophilic archaebacterium, Pyrobaculum islandicum (Geo 3) was examined for the presence of lipoquinones. Thin layer chromatographic, HPLC, UV, and mass spectroscopic analysis showed that a menaquinone was present. No evidence was found for substitution of the 2-methyl-3-polyisoprenyl-1,4-naphthoquinone ring nucleus. However, the C3 isoprenoid chain consisted of six fully saturated units, and shows that the major menaquinone in this organism corresponds to 2-methyl-3-VI,V,IV,III,II,I-dodecahydrohexaprenyl-1, 4-naphthoquinone (MK-6H₁₂).     

Structure–activity relationships in the conversion of vitamin K analogues into menaquinone-4. Substrates essential to the synthesis of menaquinone-4 in cultured human cell lines

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure–activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.     

Electron transport in a Park-Williams strain of Corynebacterium diphtheriae

1. The electron-transport mechanism was examined in the ;particulate' and ;supernatant' fractions of disintegrated cells of a Park-Williams strain of Corynebacterium diphtheriae. 2. Succinate-oxidase activity was found mainly in the ;particulate' fraction, and NADH(2) oxidase mainly in the ;supernatant', which was devoid of cytochromes and menaquinone. 3. The sum of the activities of particles and supernatant fractions, with respect to both succinate oxidase and NADH(2) oxidase, was substantially less than that of the crude cell extract from which they were obtained. Full activity was restored on recombining ;particles' and ;supernatant'. The characteristics of this reassembled system were investigated. 4. The strain of organism (CN2000) examined contained cytochromes corresponding spectroscopically to ;a', ;b' and ;c' types. All three were reduced by succinate, lactate or NADH(2); but a portion of the cytochrome b, susceptible to reduction by dithionite, could not be reduced by the substrates. 5. Triton X-100 inhibits oxidation of succinate by particulate fraction; on adding succinate, the reduction of cytochrome b is not affected but that of cytochromes a and c is delayed. 6. Irradiation at 360mmu completely destroys menaquinone in the particle fraction. Succinate oxidation is severely decreased; succinate dehydrogenase and NADH(2) oxidation are little affected. Certain menaquinones will restore succinate oxidation in the irradiated material. 7. On adding succinate to irradiated particulate material cytochrome b is partially reduced at once, but reduction of cytochromes a and c is much delayed. A portion of the cytochrome b remains not reduced, but reduction occurs rapidly on the addition of menaquinone (MK-2).     

Structure and function of a menaquinone involved in electron transport in membranes of Clostridium thermoautotrophicum and Clostridium thermoaceticum.

Clostridium thermoaceticum and Clostridium thermoautotrophicum contain the same menaquinone. Its structure, determined by thin-layer chromatography, UV absorption spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy, was found to be MK-7 (2-methyl-3-heptaprenyl-1,4-naphthoquinone). The menaquinone is located in the cytoplasmic membranes and is involved in redox reactions of two b-type cytochromes present in the clostridia. These reactions were studied with right-side-out membranes prepared from C. thermoautotrophicum by using CO as an electron donor. In intact membranes, both cytochromes were reduced, whereas after inactivation of the menaquinone by exposure of the membranes to UV irradiation, reduction of the low-potential cytochrome (Eo', -200 mV) but not of the high-potential cytochrome (Eo', -48 mV) occurred. The reduction of the high-potential cytochrome in UV-irradiated membranes was restored following the addition of oxidized menaquinone and with an excess of CO. The addition of oxidized menaquinone to reduced membranes resulted initially in a preferential oxidation of the low-potential cytochrome. The results obtained indicate that the menaquinone acts between the two b-type cytochromes in an electron transport chain.     

白色杆菌属一新种的分离鉴定及其系统发育学分析

从热带土壤中分离到一株好氧、革兰氏阳性、不产芽孢的杆状菌株F8,该菌株含有MK11为主要醌组份;细胞壁肽聚糖的氨基酸组分为2,4氨基丁酸和γ氨基丁酸等;细胞壁糖组分为鼠李糖、半乳糖和葡萄糖;DNA的G+C含量为68mol%。16SrRNA基因测序和系统发育学分析的结果表明,菌株F8与驹形白色杆菌(Leucobacter komagatae)亲缘关系最近,其16S rRNA基因的同源率为96%。两者的总DNA杂交率为62%,生理生化特征也有差异,故可把菌株F8定为一个新种,即热带白色杆菌(Leucobacter tropicalissp.nov.)。     

Menaquinone-specific prenyl reductase from the hyperthermophilic archaeon Archaeoglobus fulgidus

Four genes that encode the homologues of plant geranylgeranyl reductase were isolated from a hyperthermophilic archaeon Archaeoglobus fulgidus, which produces menaquinone with a fully saturated heptaprenyl side chain, menaquinone-7(14H). The recombinant expression of one of the homologues in Escherichia coli led to a distinct change in the quinone profile of the host cells, although the homologue is the most distantly related to the geranylgeranyl reductase. The new compounds found in the profile had successively longer elution times than those of ordinary quinones from E. coli, i.e., menaquinone-8 and ubiquinone-8, in high-performance liquid chromatography on a reversed-phase column. Structural analyses of the new compounds by electron impact-mass spectrometry indicated that their molecular masses progressively increase relative to the ordinary quinones at a rate of 2 U but that they still contain quinone head structures, strongly suggesting that the compounds are quinones with partially saturated prenyl side chains. In vitro assays with dithionite as the reducing agent showed that the prenyl reductase is highly specific for menaquinone-7, rather than ubiquinone-8 and prenyl diphosphates. This novel enzyme noncovalently binds flavin adenine dinucleotide, similar to geranylgeranyl reductase, but was not able to utilize NAD(P)H as the electron donor, unlike the plant homologue.     

NADH: quinone oxidoreductase as a site of Na+-dependent activation in the respiratory chain of marine Vibrio alginolyticus.

The site of Na+-dependent activation in the respiratory chain of the marine bacterium, Vibrio alginolyticus, was investigated. The respiratory chain system contained ubiquinones (Q), menaquinones (MK), cytochromes b(560), c(553), d(630), and o(560). The membrane-bound and partially purified NADH dehydrogenase was stimulated 2- to 3-fold by the addition of 0.2 M Na+ or K+ and no specific requirement for Na+ was observed in this reaction step. The cytochrome oxidase showed no requirement for monovalent cations. The respiratory activity (NADH oxidase) of the membrane was lost on removal of the quinones, and the reincorporation of authentic Q-10 or MK-4 restored the activity. The rate of MK-4 reduction by NADH (menaquinone reductase) as measured using MK-4 incorporated membrane was activated by Na+, but only slightly by K+. The apparent Ka for Na+ was 78 mM for both menaguinone reductase and NADH oxidase. The requirement for Na+ of menaquinone reductase was greatly reduced in the presence of 0.2 M K+. Ubiquinone reductase as measured by using Q-10 incorporated membrane was also activated more effectively by Na+ than by K+. These results strongly suggested that the site of Na+-dependent activation in the respiratory chain of marine V. alginolyticus was at the step of NADH; quinone oxidoreductase.     

Bacterial phospholipid molecular species analysis by ion-pair reversed-phase HPLC/ESI/MS

This work set out to optimize the detection and separation of several phospholipid molecular species on a reversed-phase column with the use of an electrospray ionization/mass spectrometry-compatible counter-ion. An application of this technique concerned a qualitative and quantitative analysis of bacterial membrane phospholipids extracted from Corynebacterium species strain 8. The phospholipid classes of strain 8 were identified as phosphatidylglycerol, phosphatidylinositol, diphosphatidylglycerol, and a peculiar lipid compound, acyl phosphatidylglycerol. Most of the molecular species structures were elucidated, and regarding phosphatidylglycerol, the fatty acid positions were clearly determined with the calculation of the sn-2/sn-1 intensity ratio of the fatty acyl chain fragments.     

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).     

8-OH-DPAT and MK-801 affect epileptic activity independently of vigilance

Vigilance and parallel occurrence of epileptic activity after administration of the 5-HT_1A agonist 8-OH-DPAT and the NMDA receptor antagonist MK-801 were studied in the genetic absence epilepsy model WAG/Rij rats. Spike-wave discharges (SWD) were present predominantly in passive awake and light slow wave sleep (SWS1) either in control animals or after treatments. Injection of 8-OH-DPAT (20.0 μg/rat i.c.v.) caused marked increase and MK-801 (10.0 μg/rat i.c.v.) decrease in SWD densities, thus the ratios of SWD in passive awake and in SWS1. SWD densities of MK-801 plus 8-OH-DPAT in combination were similar to those of CSF+CSF treated control rats. Both 8-OH-DPAT and MK-801 transiently increased the duration of active awake, increased latency and decreased duration of rapid eye movement (REM) sleep. 8-OH-DPAT increased the amount of SWD despite the decrease in the duration of SWS1. MK-801 decreased the amount of SWD despite the lack of significant change in duration of passive awake or SWS1. Pre-treatment with MK-801 reversed 8-OH-DPAT- induced increase in duration of SWD without any effect on 8-OH-DPAT-induced changes in sleep parameters. Our studies provide evidence that 8-OH-DPAT-induced epileptic activity is independent of its effect on sleep, and that interaction of serotonergic and glutamatergic systems plays a role in the generation of SWD, but not in the regulation of vigilance and sleep.     

Metabolic engineering of menaquinone-8 pathway of Escherichia coli as a microbial platform for vitamin K production

Menaquinone-8 (MK-8, vitamin K) is composed of a non-polar side chain and a polar head group. Escherichia coli was chosen and metabolically engineered as a microbial platform for production of MK-8. MK-8 content in E. coli was significantly enhanced by modulating two precursor pools, which supply a non-polar side chain and a polar head group, and further increased by blocking formation of the competitor ubiquinone-8 (Q-8). Overexpression of E. coli IspA, DXR, or IDI increased MK-8 content up to twofold. A similar positive effect was also observed when E. coli MenA, MenB, MenC, MenD, MenE, MenF, or UbiE was overexpressed. The Q-8-deficient ubiCA mutant enhanced MK-8 content by 30% compared to wild-type E. coli. When MenA or MenD was overexpressed, MK-8 content was enhanced fivefold compared with wild-type E. coli.     

Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.

A convenient method for the separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography (HPLC) is described. Sphingomyelin species from bovine brain and sheep and pig erythrocytes were resolved into 10-12 separate peaks on a micro -BondaPak C(18) or Nucleosil-5-C(18) reversedphase column with methanol-5 mM potassium phosphate buffer, pH 7.4, 9:1 (v/v) as the solvent. Detection was at 203-205 nm. The sphingomyelin species were primarily resolved due to specific hydrophobic interaction of their fatty acid and sphingoid chains with the alkyl ligand of the stationary phase. The retention time of the sphingomyelin species increased progressively as the number of carbon atoms in the hydrophobic chains increased in the homologous series. The presence of one double bond in the molecule reduced the retention time significantly. Introduction of a second double bond in the fatty acid side chain did not reduce the retention time to the same extent as the first double bond. The presence of a trans double bond in the sphingoid moiety increased the retention time of sphingomyelin more than did a cis double bond in the fatty acid side chain. The differential hydrophobic interaction observed between the ligand of the stationary phase and different alkyl chains of the sphingomyelin species illustrates that reversed-phase HPLC technique can be conveniently used to study the extent of relative hydrophobicity of different types of alkyl chains.-Jungalwala, F. B., V. Hayssen, J. M. Pasquini, and R. H. McCluer. Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography.     

Improved Extraction of Rice Prolamin

A considerable amount of menaquinone (MK)-4 was found in cells of a l-hydroxy-2- naphthoate-resistant mutant, strain HNA 250–15, which was derived from Flavobacterium sp. 238- 7, in which MK-6 is the major isoprenoid quinone. The MK-4 productivity was further improved by making the mutant resistant to usnic acid and menadione. The amount of MK produced by the resultant mutant, strain K₃–15, produced 125.4mg/1 of culture broth and 12.8 mg/g of dry cell weight, in the ratio of MK-4 and MK-6 of 6:1, under the optimal culture conditions in the presence of cedar wood oil.     

Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., two new members of the family Dermatophilaceae, and reclassification of Dermatophilus chelonae (Masters et al. 1995) as Austwickia chelonae gen. nov., comb. nov

Two Gram-positive bacteria, designated strains Aji5-31(T) and Ngc37-23(T), were isolated from the intestinal tracts of fishes. 16S rRNA gene sequence analysis indicated that both strains were related to the members of the family Dermatophilaceae, with 95.6-96.9% 16S rRNA gene sequence similarities. The family Dermatophilaceae contains 2 genera and 3 species: Dermatophilus congolensis, Dermatophilus chelonae and Kineosphaera limosa. However, it has been suggested that the taxonomic position of D. chelonae should be reinvestigated using a polyphasic approach, because the chemotaxonomic characteristics are not known (Stackebrandt, 2006; Stackebrandt and Schumann, 2000). Our present study revealed that strains Aji5-31(T), Ngc37-23(T) and D. chelonae NBRC 105200(T) should be separated from the other members of the family Dermatophilaceae on the basis of the following characteristics: the predominant menaquinone of strain Aji5-31(T) is MK-8(H(2)), strain Ngc37-23(T) possesses iso- branched fatty acids as major components, and the menaquinone composition of D. chelonae is MK-8(H(4)), MK-8 and MK-8(H(2)) (5 : 3 : 2, respectively). On the basis of these distinctive phenotypic characteristics and phylogenetic analysis results, it is proposed that strains Aji5-31(T) and Ngc37-23(T) be classified as two novel genera and species of the family Dermatophilaceae. The names are Mobilicoccus pelagius gen. nov., sp. nov. and Piscicoccus intestinalis gen. nov., sp. nov., and the type strains are Aji5-31(T) (=NBRC 104925(T) =DSM 22762(T)) and Ngc37-23(T) (=NBRC 104926(T) =DSM 22761(T)), respectively. In addition, D. chelonae should be reassigned to a new genus of the family Dermatophilaceae with the name Austwickia chelonae gen. nov., comb. nov.     

Interaction of lipophilic quinones with membrane fragments of Paracoccus denitrificans and Staphylococcus epidermidis.

In quinone-depleted mitochondrial and Paracoccus denitrificans membranes the quantum yield of fluorescence of ostruthin (6-geranyl-7-hydroxycoumarin) was maintained, whereas an increase in the quantum yield took place after extraction of Staphylococcus epidermidis membrane. A marked quenching effect of ubiquinone and menaquinone each with two isoprene units in the side chain on the ostruthin fluorescence was found with all types of quinone-depleted particles. When the homogues of menaquinone and ubiquinone with six isoprene units in the side chain were re-incorporated, a quenching of the ostruthin fluorescence was observed in the S. epidermidis membranes but not in those of P. denitrificans. The different behaviour of both bacterial preparations is attributable to the more specific finding of ubiquinone in the particles of P. denitrificans.     

Reconstitution of coupled fumarate respiration in liposomes by incorporating the electron transport enzymes isolated from Wolinella succinogenes.

Hydrogenase and fumarate reductase isolated from Wolinella succinogenes were incorporated into liposomes containing menaquinone. The two enzymes were found to be oriented solely to the outside of the resulting proteoliposomes. The proteoliposomes catalyzed fumarate reduction by H2 which generated an electrical proton potential (Delta(psi) = 0.19 V, negative inside) in the same direction as that generated by fumarate respiration in cells of W. succinogenes. The H+/e ratio brought about by fumarate reduction with H2 in proteoliposomes in the presence of valinomycin and external K+ was approximately 1. The same Delta(psi) and H+/e ratio was associated with the reduction of 2,3-dimethyl-1,4-naphthoquinone (DMN) by H2 in proteoliposomes containing menaquinone and hydrogenase with or without fumarate reductase. Proteoliposomes containing menaquinone and fumarate reductase with or without hydrogenase catalyzed fumarate reduction by DMNH2 which did not generate a Delta(psi). Incorporation of formate dehydrogenase together with fumarate reductase and menaquinone resulted in proteoliposomes catalyzing the reduction of fumarate or DMN by formate. Both reactions generated a Delta(psi) of 0.13 V (negative inside). The H+/e ratio of formate oxidation by menaquinone or DMN was close to 1. The results demonstrate for the first time that coupled fumarate respiration can be restored in liposomes using the well characterized electron transport enzymes isolated from W. succinogenes. The results support the view that Delta(psi) generation is coupled to menaquinone reduction by H2 or formate, but not to menaquinol oxidation by fumarate. Delta(psi) generation is probably caused by proton uptake from the cytoplasmic side of the membrane during menaquinone reduction, and by the coupled release of protons from H2 or formate oxidation on the periplasmic side. This mechanism is supported by the properties of two hydrogenase mutants of W. succinogenes which indicate that the site of quinone reduction is close to the cytoplasmic surface of the membrane.     

Chemotaxonomic differentiation of coryneform bacteria isolated from biofilters.

Coryneform bacteria that were isolated from biofilters which are used for waste gas treatment of animal-rendering plant emissions were differentiated and partially identified by using chemotaxonomic methods. On the basis of the results of a numerical analysis of whole-cell fatty acid profiles, 79 isolates were divided into two major groups; the members of the first group contained saturated and monounsaturated fatty acids, whereas the members of the second group were characterized by iso- and anteiso-branched fatty acids. Division into subclusters was based mainly on quantitative differences in fatty acid composition and was confirmed by the results obtained for additional chemical markers (e.g., respiratory quinones, mycolic acids, polar lipids, cell wall amino acids, and whole-cell sugar patterns). By combining the results obtained for chemotaxonomic analyses that were performed for strains containing saturated and monounsaturated fatty acids, we were able to identify the genus Corynebacterium (two Corynebacterium species were differentiated on the basis of the occurrence of tuberculostearic acid), the genus Gordona, and the genus Mycobacterium. Among the strains that produced iso-anteiso fatty acid patterns, one subgroup was affiliated with the "nicotianae" group of the genus Arthrobacter; however, some strains contained a new combination of chemical markers. Peptidoglycan type A4 alpha, L-Lys-Gly-L-Glu was combined with menaquinones MK-7 and MK-8, whereas peptidoglycan type A4 alpha, L-Lys-L-Glu occurred together with MK-8 and MK-9. The second subgroup was characterized by a new type B peptidoglycan and MK-11, as well as small amounts of MK-12. Differentiation that was based first on chemotaxonomy and second on physiology gave reliable results. Thus, coryneform strains with new characteristics were isolated from biofilters.     

Efficient production of α-acetolactate by whole cell catalytic transformation of fermentation-derived pyruvate

With a variety of physiological and pharmacological functions, menaquinone is an essential prenylated product that can be endogenously converted from phylloquinone (VK1) or menadione (VK3) via the expression of Homo sapiens UBIAD1 (HsUBIAD1). The methylotrophic yeast, Pichia pastoris, is an attractive expression system that has been successfully applied to the efficient expression of heterologous proteins. However, the menaquinone biosynthetic pathway has not been discovered in P. pastoris. Firstly, we constructed a novel synthetic pathway in P. pastoris for the production of menaquinone-4 (MK-4) via heterologous expression of HsUBIAD1. Then, the glyceraldehyde-3-phosphate dehydrogenase constitutive promoter (PGAP) appeared to be mostsuitable for the expression of HsUBIAD1 for various reasons. By optimizing the expression conditions of HsUBIAD1, its yield increased by 4.37 times after incubation at pH 7.0 and 24 °C for 36 h, when compared with that under the initial conditions. We found HsUBIAD1 expressed in recombinant GGU-23 has the ability to catalyze the biosynthesis of MK-4 when using VK1 and VK3 as the isopentenyl acceptor. In addition, we constructed a ribosomal DNA (rDNA)-mediated multi-copy expression vector for the fusion expression of SaGGPPS and PpIDI, and the recombinant GGU-GrIG afforded higher MK-4 production, so that it was selected as the high-yield strain. Finally, the yield of MK-4 was maximized at 0.24 mg/g DCW by improving the GGPP supply when VK3 was the isopentenyl acceptor. In this study, we constructed a novel synthetic pathway in P. pastoris for the biosynthesis of the high value-added prenylated product MK-4 through heterologous expression of HsUBIAD1 and strengthened accumulation of GGPP. This approach could be further developed and accomplished for the biosynthesis of other prenylated products, which has great significance for theoretical research and industrial application.