透明颤菌血红蛋白的表达及对基因工程菌的影响

利用已克隆的透明颤菌(Vitreoscilla)血红蛋白基因(vgb),构建了一批复制类型和抗生标记不同的vgb表达载体,并就vgb基因表达及其对几种基因工程大肠杆菌的影响进行了初步研究。实验证明vgb基因的表达具有氧调控特性,在溶氧水平下跌至20％饱和度时迅速合成。Vgb基因的表达产物(Vitreoscilla Hemoglogin,VHb)可促进青霉素酰化酶和TNF、IL-2等基因工程菌在低氧条件下细胞生长和产物表达的状况,由于vgb基因的表达降低了细胞对氧的敏感程度,可望运用它来改善发酵过程中溶氧控制裕度。这些实验结果预示着vgb基因在耗氧生物过程中,如抗生素工业和基因工程菌高密度发酵,有着良好的应用前景。     

Oscillation of NAD(P)H fluorescence in Escherichia coli culture performing dissimilative nitrate/nitrite reduction

When nitrate was added to anaerobic resting cultures of Escherichia coli, two different profiles of NAD(P)H fluorescence were observed. E. coli is known to reduce nitrate to ammonia via nitrite as an anaerobic respiration mechanism. The profile showing single-stage response corresponded to situations where the nitrite formed from nitrate reduction was immediately converted to ammonia. The other profile showing two-stage response resulted from a much slower reduction of nitrite than nitrate. Nitrite thus accumulated during the first stage and was gradually reduced to ammonia when nitrate was depleted, i.e. in the second stage. An undamped oscillation of NAD(P)H fluorescence was also observed in the cultures showing the two-stage response. The oscillation was always detected during the second stage and seldom during either the first stage or the recovered anaerobic stage (after complete nitrite reduction). It never occurred in the cultures showing the single-stage response. The period of oscillation ranged from 1 to 5min. The possibility of the common glycolytic oscillation being responsible is low, as judged from the current knowledge of the nitrate/nitrite reductases of E. coli and the observations in this study. This is the first report on the occurrence of oscillatory NAD(P)H fluorescence in E. coli.     

Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state

Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.     

Effect of Vitreoscilla hemoglobin biosynthesis in Escherichia coli on production of poly(beta-hydroxybutyrate) and fermentative parameters

In order to attain high cell density and low cost production of poly(beta-hydroxybutyrate) (PHB), the Vitreoscilla globin gene (vgb) was introduced into a novel recombinant strain, Escherichia coli VG1 (pTU14). Experiments showed that the expression of vgb was under the regulation of dissolved oxygen (DO) in broth and the introduction of vgb in VG1 (pTU14) induced the parent promotion effect on cell growth and PHB accumulation, especially under low DO conditions. Further experiments indicated that the introduction of vgb in VG1 (pTU14) not only decreased the critical oxygen concentration, but also affected the volumetric oxygen transfer coefficient of the recombinant strain.     

Oscillation of NAD(P)H fluorescence in Escherichia coli culture performing dissimilative nitrate/nitrite reduction

When nitrate was added to anaerobic resting cultures of Escherichia coli, two different profiles of NAD(P)H fluorescence were observed. E. coli is known to reduce nitrate to ammonia via nitrite as an anaerobic respiration mechanism. The profile showing single-stage response corresponded to situations where the nitrite formed from nitrate reduction was immediately converted to ammonia. The other profile showing two-stage response resulted from a much slower reduction of nitrite than nitrate. Nitrite thus accumulated during the first stage and was gradually reduced to ammonia when nitrate was depleted, i.e. in the second stage. An undamped oscillation of NAD(P)H fluorescence was also observed in the cultures showing the two-stage response. The oscillation was always detected during the second stage and seldom during either the first stage or the recovered anaerobic stage (after complete nitrite reduction). It never occurred in the cultures showing the single-stage response. The period of oscillation ranged from 1 to 5min. The possibility of the common glycolytic oscillation being responsible is low, as judged from the current knowledge of the nitrate/nitrite reductases of E. coli and the observations in this study. This is the first report on the occurrence of oscillatory NAD(P)H fluorescence in E. coli.     

Effect of culture conditions on the NADH/NAD ratio and total amounts of NAD(H) in chemostat cultures of Enterococcus faecalis NCTC 775

Abstract Enterococcus faecalis was grown in chemostat culture on various energy sources at dilution rates ranging from 0.05 h⁻¹ to 0.5 h⁻¹, under both aerobic and anaerobic conditions. NADH/NAD ratios and total nicotinamide adenine dinucleotide pool size (NAD(H)) were determined. It was found that the NADH/NAD ratio was controlled by the steady state product concentrations rather than by the degree of reduction of the energy source. Highest ratios were observed when NADH was reoxidized via ethanol formation, whereas in aerobic cultures, in which predominantly acetate was produced and oxidation of NADH occurred via the NADH oxidase, ratios were lowest. Addition of ethanol to the medium resulted in an increase of the NADH/NAD ratio, both aerobically and anaerobically. The total amount of NAD(H) was found to be influenced by the culture conditions. Under anaerobic conditions, the NADH oxidation (NAD reduction) rate appeared to correlate with the total amount of nicotinamide nucleotides. In contrast, no effect of the culture conditions on the total amount of NAD(H) was observed in aerobically grown cells.     

Neither total culture fluorescence nor intracellular fluorescence are indicative of NAD(P)H levels in Escherichia coli MG 1655

Summary The blue fluorescence emitted by microbial cells irradiated with UV light at 360 nm is usually supposed to provide a good estimate of the cell NAD(P)H content. Here we present an example of a microbial fermentation in which culture fluorescence, both in the cells and in the medium, was almost exclusively due to the presence of a fluorophore that displayed an emission spectrum very similar to that of NAD(P)H but that we show by biochemical studies to be a different compound. Our results demonstrate that studies on the redox state of cells should be based on on-line fluorescence data only after appropriate control experiments to establish a definitive correlation between fluorescence and NAD(P)H levels. Offprint requests to: J. E. Bailey     

Periodic fluctuations in oxygen consumption comparing HeLa (cancer) and CHO (non-cancer) cells and response to external NAD(P)+/NAD(P)H

Oxygen consumption in the presence of cyanide was utilized as a measure of plasma membrane electron transport in Chinese hamster ovary (CHO) and human cervical carcinoma (HeLa) cell lines. Both intact cells and isolated plasma membranes carry cyanide-insensitive NADH(P)H oxidases at their external membrane surfaces (designated ECTO-NOX proteins). Regular oscillatory patterns of oxygen consumption with period lengths characteristic of those observed for rates of NADH oxidation by ECTO-NOX proteins were observed to provide evidence for transfer of protons and electrons to reduce oxygen to water. The oscillations plus the resistance to inhibition by cyanide identify the bulk of the oxygen consumption as due to ECTO-NOX proteins. With intact CHO cells, oxygen consumption was enhanced by but not dependent upon external NAD(P)H addition. With intact HeLa cells, oxygen consumption was inhibited by both NADH and NAD⁺ as was growth. The results suggest that plasma membrane electron transport from internal donors to oxygen as an external acceptor is mediated through ECTO-NOX proteins and that electron transport to molecular oxygen may be differentially affected by external pyridine nucleotides depending on cell type.     

Dissociation of superoxide production by mitochondrial complex I from NAD(P)H redox state

The relationship between the rate of superoxide production by complex I and NAD(P)H redox state was investigated in rat skeletal muscle mitochondria. A high rate of superoxide production was observed during succinate oxidation; the rate during pyruvate oxidation was over fourfold lower. However, the NAD(P)H pool was significantly less reduced during succinate oxidation than during pyruvate oxidation. We conclude that there is no unique relationship between superoxide production by complex I and the reduction state of the NAD(P)H pool. Our data suggest that less than 10% of the superoxide originates from the flavin site during reverse electron transport from succinate.     

Crystal structure of NAD(P)H:flavin oxidoreductase from Escherichia coli.

Flavin reductases use flavins as substrates and are distinct from flavoenzymes which have tightly bound flavins. The reduced flavin can serve to reduce ferric complexes and iron proteins. In Escherichia coli, reactivation of ribonucleotide reductase is achieved by reduced flavins produced by flavin reductase. The crystal structure of E. coli flavin reductase reveals that the enzyme structure is similar to the structures of the ferredoxin reductase family of flavoproteins despite very low sequence similarities. The main difference between flavin reductase and structurally related flavoproteins is that there is no binding site for the AMP moiety of FAD. The direction of the helix in the flavin binding domain, corresponding to the phosphate binding helix in the flavoproteins, is also slightly different and less suitable for phosphate binding. Interactions for flavin substrates are instead provided by a hydrophobic isoalloxazine binding site that also contains a serine and a threonine, which form hydrogen bonds to the isoalloxazine of bound riboflavin in a substrate complex.     

WrbA from Escherichia coli and Archaeoglobus fulgidus is an NAD(P)H:quinone oxidoreductase

WrbA (tryptophan [W] repressor-binding protein) was discovered in Escherichia coli, where it was proposed to play a role in regulation of the tryptophan operon; however, this has been put in question, leaving the function unknown. Here we report a phylogenetic analysis of 30 sequences which indicated that WrbA is the prototype of a distinct family of flavoproteins which exists in a diversity of cell types across all three domains of life and includes documented NAD(P)H:quinone oxidoreductases (NQOs) from the Fungi and Viridiplantae kingdoms. Biochemical characterization of the prototypic WrbA protein from E. coli and WrbA from Archaeoglobus fulgidus, a hyperthermophilic species from the Archaea domain, shows that these enzymes have NQO activity, suggesting that this activity is a defining characteristic of the WrbA family that we designate a new type of NQO (type IV). For E. coli WrbA, the K(m)(NADH) was 14 +/- 0.43 microM and the K(m)(benzoquinone) was 5.8 +/- 0.12 microM. For A. fulgidus WrbA, the K(m)(NADH) was 19 +/- 1.7 microM and the K(m)(benzoquinone) was 37 +/- 3.6 microM. Both enzymes were found to be homodimeric by gel filtration chromatography and homotetrameric by dynamic light scattering and to contain one flavin mononucleotide molecule per monomer. The NQO activity of each enzyme is retained over a broad pH range, and apparent initial velocities indicate that maximal activities are comparable to the optimum growth temperature for the respective organisms. The results are discussed and implicate WrbA in the two-electron reduction of quinones, protecting against oxidative stress.     

Improvement of bioremediation by Pseudomonas and Burkholderia by mutants of the Vitreoscilla hemoglobin gene (vgb) integrated into their chromosomes

Using genetic engineering, the Vitreoscilla (bacterial) hemoglobin gene (vgb) was integrated stably into the chromosomes of Pseudomonas aeruginosa and Burkholderia sp. strain DNT. This was done for both wild type vgb and two site-directed mutants of vgb that produce Vitreoscilla hemoglobin (VHb) with lowered oxygen affinities; in all cases functional VHb was expressed. Similar to previous results, the wild type VHb improved growth for both species and degradation of 2,4-dinitrotoluene (Burkholderia sp.) or benzoic acid (P. aeruginosa) under both normal and low aeration conditions. Both mutant vgbs enhanced these parameters compared to wild type vgb, and the improvement was seen in both species. The enhancements were generally greater at low aeration than at normal aeration. The results demonstrate the possibility that the positive effects provided by VHb may be augmented by protein engineering.     

NAD(P)H oxidase-stimulating activity of serum from type 2 diabetic patients with retinopathy mediates enhanced endothelial expression of E-selectin

Endothelial expression of E-selectin is enhanced in diabetic patients with retinopathy, however, the underlying mechanisms are unclear. Therefore, this study was aimed to determine if endothelial expression of E-selectin is stimulated with serum from type 2 diabetic patients with retinopathy, and whether this process is related to NAD(P)H oxidase-derived oxidative stress. Serum was obtained from type 2 diabetic patients with (T2DR) or without (T2DM) retinopathy, and age-matched non-diabetic healthy person (Control). Serum was added to in vitro-grown human coronary artery endothelial cells (HCAEC), after which E-selectin expression, reactive oxygen species (ROS) production, and NAD(P)H oxidase activity were measured. Serum from T2DR induced a significantly higher expression of E-selectin than serum from T2DM and control in association with an enhanced production of ROS in HCAEC. T2DR serum enhanced E-selectin expression in a ROS-dependent manner since this process was significantly attenuated not only by tiron (1 mM), a superoxide scavenger, but also by DPI (10 micromol/L) and apocynin (100 micromol/L), inhibitors of NAD(P)H oxidase. Furthermore, the activity of NADH oxidase was markedly increased by T2DR serum, and this was accompanied by the enhanced membrane translocation of p47phox, a cytosolic subunit of NAD(P)H oxidase. These findings suggest that serum from T2DR induced up-regulation of E-selectin expression in HCAEC, and this process might be dependent on activation of endothelial NADH oxidase via an enhanced membrane translocation of p47phox.     

NAD(P)H fluorescence imaging of mitochondrial metabolism in contracting Xenopus skeletal muscle fibers: effect of oxygen availability.

The blue autofluorescence (351 nm excitation, 450 nm emission) of single skeletal muscle fibers from Xenopus was characterized to be originating from mitochondrial NAD(P)H on the basis of morphological and functional correlations. This fluorescence signal was used to estimate the oxygen availability to isolated single Xenopus muscle fibers during work level transitions by confocal microscopy. Fibers were stimulated to generate two contractile periods that were only different in the PO2 of the solution perfusing the single fibers (PO2 of 30 or 0-2 Torr; pH = 7.2). During contractions, mean cellular NAD(P)H increased significantly from rest in the low PO2 condition with the core (inner 10%) increasing to a greater extent than the periphery (outer 10%). After the cessation of work, NAD(P)H decreased in a manner consistent with oxygen tensions sufficient to oxidize the surplus NAD(P)H. In contrast, NAD(P)H decreased significantly with work in 30 Torr PO2. However, the rate of NAD(P)H oxidation was slower and significantly increased with the cessation of work in the core of the fiber compared with the peripheral region, consistent with a remaining limitation in oxygen availability. These results suggest that the blue autofluorescence signal in Xenopus skeletal muscle fibers is from mitochondrial NAD(P)H and that the rate of NAD(P)H oxidation within the cell is influenced by extracellular PO2 even at high extracellular PO2 during the contraction cycle. These results also demonstrate that although oxygen availability influences the rate of NAD(P)H oxidation, it does not prevent NAD(P)H from being oxidized through the process of oxidative phosphorylation at the onset of contractions.     

Fate of oxygen losses from Typha domingensis (Typhaceae) and Cladium jamaicense (Cyperaceae) and consequences for root metabolism

The objective of this work was to determine whether radial oxygen loss (ROL) from roots of Typha domingensis and Cladium jamaicense creates an internal oxygen deficiency or, conversely, indicates adequate internal aeration and leakage of excess oxygen to the rhizosphere. Methylene blue in agar was used to visualize the pattern of ROL from roots, and oxidation of a titanium-citrate solution was used to quantify rates of oxygen leakage. Typha's roots had a higher porosity than Cladium's and responded to flooding treatment by increasing cortical air space, particularly near the root tips. A greater oxygen release, which occurred along the subapical root axis, and an increase in rhizosphere redox potential (E(h)) over time were associated with the well-developed aerenchyma system in Typha. Typha roots, regardless of oxygen release pattern, showed low or undetectable alcohol dehydrogenase (ADH) activity or ethanol concentrations, indicating that ROL did not cause internal deficiencies. Cladium roots also released oxygen, but this loss primarily occurred at the root tips and was accompanied by increased root ADH activity and ethanol concentrations. These results support the hypothesis that oxygen release by Cladium is accompanied by internal deficiencies of oxygen sufficient to stimulate alcoholic fermentation and helps explain Cladium's lesser flood tolerance in comparison with Typha.     

Component D of chicken hemoglobin and the hemoglobin of the embryonic Tammar wallaby (Macropus eugenii) self-associate upon deoxygenation: Effect on oxygen binding

Extensive measurements of oxygen binding by some vertebrate hemoglobins (Hbs) have suggested an unusually high degree of cooperativity with reported Hill coefficients, n(H), greater than 4.0. We have reexamined this possibility of "super-cooperativity" with chicken Hb components A (alpha(A) (2)beta(2)) and D (alpha(D) (2)beta(2)). Prior studies have shown that component D but not A self-associates to dimers of tetramers upon deoxygenation. This self-association is reflected in the oxygen equilibrium of Hb D which shows a maximal n(H), greater than 4.0 at approximately 4 mM heme concentration. In contrast, component A has maximal n(H) value below 3. The value of the maximal n(H) for Hb D increases linearly with the fraction of octamer present in the deoxy Hb. We anticipate that deoxygenation-dependent self-association will be shown to be a general property of Hb D from birds and reptiles. Neither oxygen equilibria nor sedimentation measurements show any evidence that components A and D interact to form a complex when deoxygenated. We have also reexamined the oxygen equilibria of Hbs of an embryonic marsupial, the wallaby. The equilibria in red cells have been reported to have Hill coefficients as high as 5-6. Although our oxygen equilibrium measurements of solutions of unfractionated wallaby Hb at a concentration of approximately 1 mM show no n(H) values greater than approximately 3.0, sedimentation velocity measurements provide clear evidence for deoxygenation-dependent self-association.     

Spectral characterization of NAD(P)H fluorescence in intact isolated chloroplasts and leaves: Effect of chlorophyll concentration on reabsorption of blue-green fluorescence

In the present communication we report a spectral analysis of the blue-green fluorescence related to changes in NAD(P) redox state in chloroplasts and leaves. To assess the contribution of reabsorption and the inner filter effect, we compared transmission and fluorescence at different chloroplast concentrations, and showed that reabsorption by the photosynthetic pigments (chlorophylls and carotenoids) was at the origin of the two peaks in the emission spectrum in vivo. The absence of potential green-emitting fluorophores in chloroplasts was determined by measuring variable and time-resolved fluorescence at different wavelengths. We defined the conditions which optimize the UV-excited blue-green fluorescence signal dependent on NAD(P)H, and we present an example of monitoring of NAD(P)H fluorescence in intact leaves.     

FAD is a preferred substrate and an inhibitor of Escherichia coli general NAD(P)H:flavin oxidoreductase

Escherichia coli general NAD(P)H:flavin oxidoreductase (Fre) does not have a bound flavin cofactor; its flavin substrates (riboflavin, FMN, and FAD) are believed to bind to it mainly through the isoalloxazine ring. This interaction was real for riboflavin and FMN, but not for FAD, which bound to Fre much tighter than FMN or riboflavin. Computer simulations of Fre.FAD and Fre.FMN complexes showed that FAD adopted an unusual bent conformation, allowing its ribityl side chain and ADP moiety to form an additional 3.28 H-bonds on average with amino acid residues located in the loop connecting Fbeta5 and Falpha1 of the flavin-binding domain and at the proposed NAD(P)H-binding site. Experimental data supported the overlapping binding sites of FAD and NAD(P)H. AMP, a known competitive inhibitor with respect to NAD(P)H, decreased the affinity of Fre for FAD. FAD behaved as a mixed-type inhibitor with respect to NADPH. The overlapped binding offers a plausible explanation for the large K(m) values of Fre for NADH and NADPH when FAD is the electron acceptor. Although Fre reduces FMN faster than it reduces FAD, it preferentially reduces FAD when both FMN and FAD are present. Our data suggest that FAD is a preferred substrate and an inhibitor, suppressing the activities of Fre at low NADH concentrations.     

Long‐term ammonium nutrition of Arabidopsis increases the extrachloroplastic NAD(P)H/NAD(P)+ ratio and mitochondrial reactive oxygen species level in leaves but does not impair photosynthetic capacity

Ammonium nutrition has been suggested to be associated with alterations in the oxidation‐reduction state of leaf cells. Herein, we show that ammonium nutrition in Arabidopsis thaliana increases leaf NAD(P)H/NAD(P)⁺ ratio, reactive oxygen species content and accumulation of biomolecules oxidized by free radicals. We used the method of rapid fractionation of protoplasts to analyse which cellular compartments were over‐reduced under ammonium supply and revealed that observed changes in NAD(P)H/NAD(P)⁺ ratio involved only the extrachloroplastic fraction. We also showed that ammonium nutrition changes mitochondrial electron transport chain activity, increasing mitochondrial reactive oxygen species production. Our results indicate that the functional impairment associated with ammonium nutrition is mainly associated with redox reactions outside the chloroplast.