Comparison of composition-gradient sedimentation equilibrium and composition-gradient static light scattering as techniques for quantitative characterization of biomolecular interactions: A case study.

The equilibrium hetero-association of NADH oxidase and peroxiredoxin was characterized by means of independently conducted measurements of composition-gradient sedimentation equilibrium and composition-gradient static light scattering. Results obtained from both experiments were quantitatively accounted for by a model according to which a dimer of NADH oxidase forms a 1:1 equilibrium complex with a decamer of peroxiredoxin under the conditions of these experiments. The best-fit equilibrium constants for heteroassociation of the two proteins obtained from the two measurements were found to be identical to well within the uncertainty of estimate of each of the two methods. The relative virtues of each of the methods are discussed.

Purified thioredoxin reductase from O2-sensitive Bifidobacterium bifidum degrades H2O2 by interacting with alkyl hydroperoxide reductase.

Bifidobacterium is beneficial for host health and exhibits different O2 sensitivity levels among species or strains via unknown mechanisms. Bifidobacterium bifidum JCM1255T, a type species of Bifidobacterium, is an O2-sensitive bacterium that can grow under low-O2 (5%) conditions, and the growth of this species is inhibited under high-O2 conditions (10% ∼) with accumulation of H2O2. We previously reported that NADH or NAD(P)H oxidase-active fractions were detected during purification using microaerobically grown B. bifidum cells, and the active enzyme was purified from the NADH oxidase-active fraction. The purified enzyme was identified as b-type dihydroorotate dehydrogenase (DHODb) and characterized as a dominant H2O2 producer in B. bifidum. In this study, we performed further purification of the enzyme from the NAD(P)H oxidase-active fraction and characterized the purified enzyme as a part of the H2O2 degradation system in B. bifidum. This purified enzyme was identified as thioredoxin reductase (TrxR); the NAD(P)H oxidase activity of this enzyme was not expressed in anaerobically grown B. bifidum, and mRNA expression was induced by O2 exposure. Furthermore, the purified B. bifidum TrxR interacted with recombinant alkyl hydroperoxide reductase (rAhpC) and exhibited NAD(P)H peroxidase activity. These results suggest that TrxR responds to O2 and protects B. bifidum from oxidative stress by degrading H2O2 via the TrxR-AhpC system.

Cucumis sativus secretes 4'-ketoriboflavin under iron-deficient conditions.

A new compound in cucumber, Cucumis sativus, nutrient solution that appears under iron-deficient conditions, but not under ordinary culture conditions, has been revealed by HPLC analysis. The chemical structure of this compound was identified using LC-MS and NMR techniques as that of 4'-ketoriboflavin. This is the first report to show that 4'-ketoriboflavin can be found in metabolites from organisms.

Adaptive response of Amphibacillus xylanus to normal aerobic and forced oxidative stress conditions.

Amphibacillus xylanus grows at the same rate and with the same cell yield under aerobic and anaerobic conditions. Under aerobic conditions, it exhibits vigorous oxygen consumption in spite of lacking a respiratory system and haem catalase. To understand the adaptive response of A. xylanus to oxidative stresses, a genomic analysis of A. xylanus was conducted. The analysis showed that A. xylanus has the genes of four metabolic systems: two pyruvate metabolic pathways, a glycolytic metabolic pathway and an NADH oxidase (Nox)-AhpC (Prx) system. A transcriptional study confirmed that A. xylanus has these metabolic systems. Moreover, genomic analysis revealed the presence of two genes for NADH oxidase (nox1 and nox2), both of which were identified in the transcriptional analysis. The nox1 gene in A. xylanus was highly expressed under normal aerobic conditions but that of nox2 was not. A purification study of NADH oxidases indicated that the gene product of nox1 is a primary metabolic enzyme responsible for metabolism of both oxygen and reactive oxygen species. A. xylanus was successfully grown under forced oxidative stress conditions such as 0.1 mM H2O2, 0.3 mM paraquat and 80 % oxygen. Proteomic analysis revealed that manganese SOD, Prx, pyruvate dehydrogenase complex E1 and E3 components, and riboflavin synthase ß-chain are induced under normal aerobic conditions, and the other proteins except the five aerobically induced proteins were not induced under forced oxidative stress conditions. Taken together, the present findings indicate that A. xylanus has a unique defence system against forced oxidative stress.

Purification and characterization of oxygen-inducible haem catalase from oxygen-tolerant Bifidobacterium asteroides.

Bifidobacterium asteroides, originally isolated from honeybee intestine, was found to grow under 20% O(2) conditions in liquid shaking culture using MRS broth. Catalase activity was detected only in cells that were exposed to O(2) and grown in medium containing a haem source, and these cells showed higher viability on exposure to H(2)O(2). Passage through multiple column chromatography steps enabled purification of the active protein, which was identified as a homologue of haem catalase on the basis of its N-terminal sequence. The enzyme is a homodimer composed of a subunit with a molecular mass of 55 kDa, and the absorption spectrum shows the typical profile of bacterial haem catalase. A gene encoding haem catalase, which has an amino acid sequence coinciding with the N-terminal amino acid sequence of the purified protein, was found in the draft genome sequence data of B. asteroides. Expression of the katA gene was induced in response to O(2) exposure. The haem catalase from B. asteroides shows about 70-80% identity with those from lactobacilli and other lactic acid bacteria, and no homologues were found in other bifidobacterial genomes.

Lactobacillus ozensis sp. nov., isolated from mountain flowers.

Five strains (Mizu2-1(T), Gon2-7, Koba6-1, Koyu2-2 and Miya2-2) of lactic acid bacteria (LAB) were isolated from flowers in Oze National Park, Japan, using anaerobic cultivation. The five isolates were found to share identical 16S rRNA gene sequences. The isolates exhibited low levels of 16S rRNA gene sequence similarity to known LAB; the closest recognized relatives of strain Mizu2-1(T) were the type strains of Lactobacillus kunkeei (94.9 %), Lactobacillus kefiri (94.1 %) and Lactobacillus buchneri (93.9 %). Comparative analyses of rpoA and pheS gene sequences demonstrated that the novel isolates did not show significant relationships to other Lactobacillus species. The strains were Gram-stain-positive, catalase-negative and heterofermentative. Anaerobic growth was better than aerobic growth. The isolates utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose and fructose. On the basis of phenotypic characteristics and phylogenetic data, the isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus ozensis sp. nov. is proposed. The type strain is Mizu2-1(T) ( = JCM 17196(T)  = DSM 23829(T)).

Lactobacillus floricola sp. nov., lactic acid bacteria isolated from mountain flowers.

Five strains (Ryu1-2(T), Gon2-9, Ryu4-3, Nog8-1 and Aza1-1) of lactic acid bacteria were isolated from flowers in mountainous areas in Japan, Oze National Park, Iizuna mountain and the Nikko area. The five isolates were found to share almost identical (99.6-100 % similar) 16S rRNA gene sequences and were therefore deemed to belong to the same species. These isolates exhibited low levels of 16S rRNA gene sequence similarity to known lactic acid bacteria; the closest recognized relatives to strain Ryu1-2(T) were the type strains of Lactobacillus hilgardii (92.8 % similarity), Lactobacillus kefiri (92.7 %), Lactobacillus composti (92.6 %) and Lactobacillus buchneri (92.4 %). Comparative analyses of rpoA and pheS gene sequences demonstrated that the novel isolates did not show significant relationships to other Lactobacillus species. The strains were Gram-stain-positive, catalase-negative and homofermentative. The isolates utilized a narrow range of carbohydrates as sources of carbon and energy, including glucose and fructose. On the basis of phenotypic characteristics and phylogenetic data, these isolates represent a novel species of the genus Lactobacillus, for which the name Lactobacillus floricola sp. nov. is proposed. The type strain is Ryu1-2(T) ( = NRIC 0774(T)  = JCM 16512(T)  = DSM 23037(T)).

Synechocystis ferredoxin-NADP(+) oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin.

We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP(+) oxidoreductase (FNR( S )). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR( S ) may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR( S ) in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR( S ) is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR( S ) was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.

Crystallization and preliminary X-ray analysis of NADH:rubredoxin oxidoreductase from Clostridium acetobutylicum.

NADH:rubredoxin oxidoreductase (NROR), an O(2)-inducible protein, is a versatile electron donor for scavengers of O(2) and reactive oxygen species (ROS) in Clostridium acetobutylicum. Recombinant NROR was overexpressed in Escherichia coli and purified to homogeneity; it was subsequently crystallized using the sitting-drop vapour-diffusion method at 293 K. Preliminary crystallographic analysis revealed that the crystals belonged to space group P4(1)22 or P4(3)22, with unit-cell parameters a = b = 98.6, c = 88.3 A, and diffracted to 2.1 A resolution. Assuming that the crystals contained one molecule per asymmetric unit, the Matthews coefficient was calculated to be 2.7 A(3) Da(-1) and the solvent content to be 54.1%.

Escherichia coli ferredoxin-NADP+ reductase and oxygen-insensitive nitroreductase are capable of functioning as ferric reductase and of driving the Fenton reaction.

Two free flavin-independent enzymes were purified by detecting the NAD(P)H oxidation in the presence of Fe(III)-EDTA and t-butyl hydroperoxide from E. coli. The enzyme that requires NADH or NADPH as an electron donor was a 28 kDa protein, and N-terminal sequencing revealed it to be oxygen-insensitive nitroreductase (NfnB). The second enzyme that requires NADPH as an electron donor was a 30 kDa protein, and N-terminal sequencing revealed it to be ferredoxin-NADP(+) reductase (Fpr). The chemical stoichiometry of the Fenton activities of both NfnB and Fpr in the presence of Fe(III)-EDTA, NAD(P)H and hydrogen peroxide was investigated. Both enzymes showed a one-electron reduction in the reaction forming hydroxyl radical from hydrogen peroxide. Also, the observed Fenton activities of both enzymes in the presence of synthetic chelate iron compounds were higher than their activities in the presence of natural chelate iron compounds. When the Fenton reaction occurs, the ferric iron must be reduced to ferrous iron. The ferric reductase activities of both NfnB and Fpr occurred with synthetic chelate iron compounds. Unlike NfnB, Fpr also showed the ferric reductase activity on an iron storage protein, ferritin, and various natural iron chelate compounds including siderophore. The Fenton and ferric reductase reactions of both NfnB and Fpr occurred in the absence of free flavin. Although the k(cat)/K(m) value of NfnB for Fe(III)-EDTA was not affected by free flavin, the k(cat)/K(m) value of Fpr for Fe(III)-EDTA was 12-times greater in the presence of free FAD than in the absence of free FAD.

Chlorella vulgaris aldehyde reductase is capable of functioning as ferric reductase and of driving the fenton reaction in the presence of free flavin.

The free flavin-dependent Fenton reaction was detected in cell-free extracts of Chlorella. The corresponding enzyme was purified to homogeneity, and its N-terminal sequence was highly homologous to those of aldo-keto reductase family enzymes. The purified enzyme displayed aldehyde reductase activity in the presence of NADPH. Additionally, it showed ferric reductase activity and drove the Fenton reaction in the presence of free FAD and NADH.

Esterification of side-chain oxysterols by lysosomal phospholipase A2.

Side-chain oxysterols produced from cholesterol either enzymatically or non-enzymatically show various bioactivities. Lecithin-cholesterol acyltransferase (LCAT) esterifies the C3-hydroxyl group of these sterols as well as cholesterol. Lysosomal phospholipase A2 (LPLA2) is related to LCAT but does not catalyze esterification of cholesterol. First, esterification of side-chain oxysterols by LPLA2 was investigated using recombinant mouse LPLA2 and dioleoyl-PC/sulfatide/oxysterol liposomes under acidic conditions. TLC and LC-MS/MS showed that the C3 and C27-hydroxyl groups of 27-hydroxycholesterol could be individually esterified by LPLA2 to form a monoester with the C27-hydroxyl preference. Cholesterol did not inhibit this reaction. Also, LPLA2 esterified other side-chain oxysterols. Their esterifications by mouse serum containing LCAT supported the idea that their esterifications by LPLA2 occur at the C3-hydroxyl group. N-acetylsphingosine (NAS) acting as an acyl acceptor in LPLA2 transacylation inhibited the side-chain oxysterol esterification by LPLA2. This suggests a competition between hydroxycholesterol and NAS on the acyl-LPLA2 intermediate formed during the reaction. Raising cationic amphiphilic drug concentration or ionic strength in the reaction mixture evoked a reduction of the side-chain oxysterol esterification by LPLA2. This indicates that the esterification could progress via an interfacial interaction of LPLA2 with the lipid membrane surface through an electrostatic interaction. The docking model of acyl-LPLA2 intermediate and side-chain oxysterol provided new insight to elucidate the transacylation mechanism of sterols by LPLA2. Finally, exogenous 25-hydroxycholesterol esterification within alveolar macrophages prepared from wild-type mice was significantly higher than that from LPLA2 deficient mice. This suggests that there is an esterification pathway of side-chain oxysterols via LPLA2.

Free flavins accelerate release of ferrous iron from iron storage proteins by both free flavin-dependent and -independent ferric reductases in Escherichia coli.

Ferredoxin NADP+ oxidoreductase (Fpr) and oxygen-insensitive NAD(P)H nitroreductase (NfnB) are purified from Escherichia coli JM109 (E. coli JM109) as a predominant free flavin-independent ferric reductase. In the present study, we prepared natural iron storage proteins, E. coli ferritin A (FtnA) and bacterioferritin (Bfr), to show the effective ferrous iron release from these proteins by Fpr and NfnB in the presence of free flavins. Fpr and NfnB showed flavin reductase activity for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and riboflavin, and their ferrous iron release activities were positively associated with the catalytic efficiencies (kcat/Km) for individual flavins. The ferrous iron release activity of E. coli cell-free extracts was affected by flavin reductase activity of the extracts. The Butyl TOYOPEARL column chromatography of the extracts, on the basis of NAD(P)H-dependent flavin reductase activity, resulted in the separation of six active fractions containing Fpr, NfnB, NAD(P)H-quinone oxidoreductase (QOR), flavin reductase (Fre) or alkyl hydroperoxide reductase subunit F (AhpF) as major components. Like Fpr and NfnB, recombinant QOR, Fre, and AhpF showed flavin reductase activity and ferrous iron release activity in the presence of free flavins, indicating an association of flavin reductase activity with ferrous iron releasing activity. Taken together, both free flavin-dependent and free flavin-independent ferric reductases in E. coli require free flavins to mediate an electron transfer from NAD(P)H to ferric iron in the iron storage proteins for the effective ferrous iron release.

Isolation of lactic acid bacteria capable of reducing environmental alkyl and fatty acid hydroperoxides, and the effect of their oral administration on oxidative-stressed nematodes and rats.

Reinforcement of the hydroperoxide-eliminating activity in the small and large intestines should prevent associated diseases. We previously isolated a lactic acid bacterium, Pediococcus pentosaceus Be1 that facilitates a 2-electron reduction of hydrogen peroxide to water. In this study, we successfully isolated an alternative lactic acid bacterium, Lactobacillus plantarum P1-2, that can efficiently reduce environmental alkyl hydroperoxides and fatty acid hydroperoxides to their corresponding hydroxyl derivatives through a 2-electron reduction. Each strain exhibited a wide concentration range with regard to the environmental reducing activity for each hydroperoxide. Given this, the two lactic acid bacteria were orally administered to an oxygen-sensitive short-lived nematode mutant, and this resulted in a significant expansion of its lifespan. This observation suggests that P. pentosaceus Be1 and L. plantarum P1-2 inhibit internal oxidative stress. To determine the specific organs involved in this response, we performed a similar experiment in rats, involving induced lipid peroxidation by iron-overloading. We observed that only L. plantarum P1-2 inhibited colonic mucosa lipid peroxidation in rats with induced oxidative stress.

b-type dihydroorotate dehydrogenase is purified as a H2O2-forming NADH oxidase from Bifidobacterium bifidum.

Our previous report showed the existence of microaerophilic Bifidobacterium species that can grow well under aerobic conditions rather than anoxic conditions in a liquid shaking culture. The difference in the aerobic growth properties between the O(2)-sensitive and microaerophilic species is due to the existence of a system to produce H(2)O(2) in the growth medium. In this study, we purified and characterized the NADH oxidase that is considered to be a key enzyme in the production of H(2)O(2). Bifidobacterium bifidum, an O(2)-sensitive bacterium and the type species of the genus Bifidobacterium, possessed one dominant active fraction of NADH oxidase and a minor active fraction of NAD(P)H oxidase activity detected in the first step of column chromatography for purification of the enzyme. The dominant active fraction was further purified and determined from its N-terminal sequence to be a homologue of b-type dihydroorotate dehydrogenase (DHOD), composed of PyrK (31 kDa) and PyrDb (34 kDa) subunits. The genes that encode PyrK and PryDb are tandemly located within an operon structure. The purified enzyme was found to be a heterotetramer showing the typical spectrum of a flavoprotein, and flavin mononucleotide and flavin adenine dinucleotide were identified as cofactors. The purified enzyme was characterized as the enzyme that catalyzes the DHOD reaction and also catalyzes a H(2)O(2)-forming NADH oxidase reaction in the presence of O(2). The kinetic parameters suggested that the enzyme could be involved in H(2)O(2) production in highly aerated environments.

O2 and reactive oxygen species detoxification complex, composed of O2-responsive NADH:rubredoxin oxidoreductase-flavoprotein A2-desulfoferrodoxin operon enzymes, rubperoxin, and rubredoxin, in Clostridium acetobutylicum.

Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O(2)-flow culture conditions, where the cells consume O(2) proficiently. An O(2)-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacterized flavoprotein A2 (FprA2), and the predicted superoxide reductase desulfoferrodoxin (Dsr), has been proposed to participate in defense against O(2) stress. To functionally characterize these proteins, native NROR from C. acetobutylicum, recombinant NROR (rNROR), FprA2, Dsr, and rubredoxin (Rd) expressed in Escherichia coli were purified. Purified native NROR and rNROR both exhibited weak H(2)O(2)-forming NADH oxidase activity that was slightly activated by Rd. A mixture of NROR, Rd, and FprA2 functions as an efficient H(2)O-forming NADH oxidase with a high affinity for O(2) (the K(m) for O(2) is 2.9 +/- 0.4 microM). A mixture of NROR, Rd, and Dsr functions as an NADH-dependent O(2)(-) reductase. A mixture of NROR, Rd, and rubperoxin (Rpr, a rubrerythrin homologue) functions as an inefficient H(2)O-forming NADH oxidase but an efficient NADH peroxidase with a low affinity for O(2) and a high affinity for H(2)O(2) (the K(m)s for O(2) and H(2)O(2) are 303 +/- 39 microM and

Expression pattern of a chloroplast NADPH-dependent thioredoxin reductase in Chlorella vulgaris during hardening and its interaction with 2-Cys peroxiredoxin.

A chloroplastic NADPH-dependent thioredoxin reductase gene was identified from Chlorella vulgaris and designated CvNTRC. Mature CvNTRC protein (mCvNTRC) was expressed in Escherichia coli, and it showed both NADPH-dependent thioredoxin reductase (NTR) and thioredoxin (Trx)-like dithiol-disulfide oxidoreductase activities. The transcript of CvNTRC increased throughout 24-h hardening, whereas the encoded protein amount and total NTR activity decreased once and then increased during hardening. By in vitro pull-down assay, a 21.2-kDa protein bound to mCvNTRC was isolated and identified as a 2-Cys peroxiredoxin (2-Cys Prx) based on the N-terminal sequence. These data suggest that CvNTRC is maintained at a constant level during hardening and functions as an antioxidant with 2-Cys Prx in the acquisition of freezing tolerance of Chlorella.

Taxonomical and physiological comparisons of the three species of the genus Amphibacillus.

Amphibacillus is a genus for Gram-positive, spore-forming, rod-shaped, facultatively anaerobic bacteria with low-G+C content of DNA, established by Niimura et al. in 1990. Amphibacillus xylanus, the type species of the genus, grows well under both strictly anaerobic and aerobic conditions in spite of lacking any isoprenoid quinones, cytochromes, and catalase. Amphibacillus fermentum and Amphibacillus tropicus were later proposed by Zhilina et al. in 2001 for the isolates from a soda lake. In this paper, we revealed the latter two species also lacked isoprenoid quinones, cytochrome and catalase, and that they grew well under strictly anaerobic and aerobic conditions. The consistent growth of A. xylanus under both conditions is due to the presence of anaerobic and aerobic pathways for glucose metabolism in the organism. Although A. fermentum and A. tropicus are supposed to have a side enzymatic pyruvate pathway to produce lactate under both conditions, the two species have two major pyruvate metabolic pathways as observed in A. xylanus. Analysis data indicated that NADH formed both by the aerobic pyruvate pathway and by the glycolytic pathway was re-oxidized by the NADH oxidase in A. fermentum and A. tropicus as well as A. xylanus, and furthermore that the NADH oxidase-Prx (AhpC) system, i.e., NADH oxidase scavenging hydrogen peroxide with Prx, also functions in A. tropicus as observed with A. xylanus. Not only the taxonomical character of the genus Amphibacillus but also the growth characterization based on the two metabolic pathways and unique oxygen metabolism are distinctive in those traits from other facultative anaerobes.

The NADH oxidase-Prx system in Amphibacillus xylanus.

Amphibacillus NADH oxidase belongs to a growing new family of peroxiredoxin-linked oxidoreductases including alkyl hydroperoxide reductase F (AhpF). Like AhpF it displays extremely high hydroperoxide reductase activity in the presence of a Prx, thus making up the NADH oxidase-Prx system. The NADH oxidase primarily catalyzes the reduction of oxygen by NADH to form H2O2, while the Prx immediately reduces H2O2 (or ROOH) to water (or ROH). Consequently, the NADH oxidase-Prx system catalyzes the reduction of both oxygen and hydrogen peroxide to water with NADH as the preferred electron donor. The NADH oxidase-Prx system is widely distributed in aerobically growing bacteria lacking a respiratory chain and catalase, and plays an important role not only in scavenging hydroperoxides but also in regenerating NAD in these bacteria.

O2-inducible H2O2-forming NADPH oxidase is responsible for the hyper O2 sensitivity of Bifidobacterium longum subsp. infantis.

Bifidobacteria are beneficial anaerobes, and their O2 sensitivity levels differ among species as a function of unknown molecular mechanisms. Bifidobacterium longum subspecies infantis (B. infantis), a predominant colonizer of the gastrointestinal tract of infants, showed a hyper O2-sensitive growth profile with accompanying a production of H2O2. In this study, we characterized an NADPH oxidase as a key enzyme responsible for this microbe's hyper O2 sensitivity. A dominant active elution peak of H2O2-forming NADPH oxidase activity was detected in the first step of column chromatography, and the purified NADPH oxidase (NPOX) was identified as a homolog of nitroreductase family proteins. The introduction of the gene encoding B. infantis NPOX (npoxA) into O2-tolerant Bifidobacterium minimum made the strain O2 sensitive and allowed it to produce H2O2. Knockout of the npoxA gene in B. infantis decreased the production of H2O2 and mitigated its B. infantis hyper O2 sensitivity. A transcript of B. infantis npoxA is induced by O2, suggesting that the aerobic production of toxic H2O2 is functionally conserved in B. infantis.