转录调控因子Rex的功能及调节机制研究进展

转录因子Rex是一种广泛存在于革兰氏阳性菌,能够与NADH或者NAD⁺直接结合响应胞内NADH/NAD⁺的氧化还原传感器,与靶基因的结合可调节细胞内的多种生理代谢。NAD（H）是调节细胞能量代谢的必需辅酶,显示微生物细胞内的氧化还原状态。研究发现Rex的调节活性与细胞内NADH/NAD⁺比率相关。需氧和厌氧菌属中Rex单体和复合物晶体结构的解析揭示了Rex、NADH/NAD⁺和靶基因间的作用关系及调控机制。通过比较分析了不同菌株中Rex单体和复合物的晶体蛋白结构,并揭示了NADH/NAD⁺对Rex调控活性的影响,进一步解析了Rex与碳和能量代谢、厌氧代谢、发酵、生物膜等之间的联系,并展望了Rex的研究和应用方向。     

Transcriptional regulation of central carbon and energy metabolism in bacteria by redox-responsive repressor Rex

Redox-sensing repressor Rex was previously implicated in the control of anaerobic respiration in response to the cellular NADH/NAD(+) levels in gram-positive bacteria. We utilized the comparative genomics approach to infer candidate Rex-binding DNA motifs and assess the Rex regulon content in 119 genomes from 11 taxonomic groups. Both DNA-binding and NAD-sensing domains are broadly conserved in Rex orthologs identified in the phyla Firmicutes, Thermotogales, Actinobacteria, Chloroflexi, Deinococcus-Thermus, and Proteobacteria. The identified DNA-binding motifs showed significant conservation in these species, with the only exception detected in Clostridia, where the Rex motif deviates in two positions from the generalized consensus, TTGTGAANNNNTTCACAA. Comparative analysis of candidate Rex sites revealed remarkable variations in functional repertoires of candidate Rex-regulated genes in various microorganisms. Most of the reconstructed regulatory interactions are lineage specific, suggesting frequent events of gain and loss of regulator binding sites in the evolution of Rex regulons. We identified more than 50 novel Rex-regulated operons encoding functions that are essential for resumption of the NADH:NAD(+) balance. The novel functional role of Rex in the control of the central carbon metabolism and hydrogen production genes was validated by in vitro DNA binding assays using the TM0169 protein in the hydrogen-producing bacterium Thermotoga maritima.     

Association and redox properties of the putidaredoxin reductase-nicotinamide adenine dinucleotide complex

Putidaredoxin reductase (PdR) is the flavin protein that carries out the first electron transfer involved in the cytochrome P450cam catalytic cycle. In PdR, the flavin adenine dinucleotide (FAD/FADH2) redox center acts as a transformer by accepting two electrons from soluble nicotinamide adenine dinucleotide (NAD+/NADH) and donating them in two separate, one-electron-transfer steps to the iron-sulfur protein putidaredoxin (Pdx). PdR, like the two more intensively studied monoflavin reductases, adrenodoxin reductase (AdR) and ferredoxin-NADP+ reductase (FNR), has no other active redox moieties (e.g., sulfhydryl groups) and can exist in three different oxidation states: (i) oxidized quinone, (ii) one-electron reduced semiquinone (stable neutral species (blue) or unstable radical anion (red)), and (iii) two-electron fully reduced hydroquinone. Here, we present reduction potential measurements for PdR in support of a thermodynamic model for the modulation of equilibria among the redox components in this initial electron-transfer step of the P450 cycle. A spectroelectrochemical technique was used to measure the midpoint oxidation-reduction potential of PdR that had been carefully purified of all residual NAD+, E0' = -369 +/- 10 mV at pH 7.6, which is more negative than previously reported and more negative than the pyridine nucleotide NADH/NAD+ (-330 mV). After addition of NAD+, the formation of the oxidized reductase-oxidized pyridine nucleotide complex was followed by the two-electron-transfer redox reaction, PdRox:NAD+ + 2e- --> PdRrd:NAD+, when the electrode potential was lowered. The midpoint potential was a hyperbolic function of increasing NAD+ concentration, such that at concentrations of pyridine nucleotide typically found in an intracellular environment, the midpoint potential would be E0' = -230 +/- 10 mV, thereby providing the thermodynamically favorable redox equilibria that enables electron transfer from NADH. This thermodynamic control of electron transfer is a shared mechanistic feature with the adrenodoxin P450 and photosynthetic electron-transfer systems but is different from the kinetic control mechanisms in the microsomal P450 systems where multiple reaction pathways draw on reducing power held by NADPH-cytochrome P450 reductase. The redox measurements were combined with protein fluorescence quenching of NAD+ binding to oxidized PdR to establish that the PdRox:NAD+ complex (KD = 230 microM) is about 5 orders of magnitude weaker than PdRrd:NAD+ binding. These results are integrated with known structural and kinetic information for PdR, as well as for AdR and FNR, in support of a compulsory ordered pathway to describe the electron-transfer processes catalyzed by all three reductases.     

ATPase activity of isolated plasma membrane vesicles of leaves of Elodea as affected by thiol reagents and NADH/NAD+ ratio

The goal of this study was to test the hypothesis that the plasma membrane-bound ATPase activity is influenced by the redox poise of the cytoplasm. Purified plasma membrane vesicles from leaves of Elodea canadensis Michx. and E. nuttallii (Planch.) St. John were isolated using an aqueous polymer two-phase batch procedure. The distribution of marker enzyme activities confirmed the plasma membrane origin of the vesicles. The vesicles exhibited NADH-ferricyanide reductase activity, indicating the presence of a redox chain in the plasma membrane. The K⁺, Mg²⁺-ATPase activity associated with these vesicles was inhibited by the sulfhydryl reagents N-ethylmaleimide and glutathione (GSSG). Furthermore the activity was inhibited by NAD⁺. This inhibition by NAD⁺ was relieved by increasing the NADH/NAD⁺ ratio. The possibility that the ATPase activity is regulated by the cytoplasmic NAD(P)H/ NAD(P)⁺ ratio is discussed, as well as the role of a plasma membrane-bound redox chain.     

Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor

NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.     

Studies of coenzyme binding to rabbit muscle glyceraldehyde-3-phoshate dehydrogenase.

The binding of NAD+, NADH and adenosine diphosphoribose (Ado-PP-Rib) to a stable, highly active and nucleotide-free preparation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) has been studied. All three nucleotides quench the protein fluorescence to the same extent when they bind to the enzyme, and this property has been used to measure the dissociation constants for the two high-affinity binding sites for the nucleotides. The results indicate negative interactions between, or non-identify of, these two binding sites, to which NAD+ and NADH bind with similar affinity. The binding of NAD+ to the enzyme has been studied by spectrophotometric titrations at 360 nm. It appears that the binding of NAD+ to each of the four subunits of the enzyme contributes equally to the intensity of this 'Racker' band. The dissociation constants associated with the binding of the third and fourth molecules of NAD+ estimated from such titrations confirm some previous estimates. The binding of NADH to the enzyme causes a decrease of intensity of the absorbance of the coenzyme at 340 nm, and the dissociation constants for binding of the third and fourth molecules of NADH have been estimated from spectrophotometric titrations. They are the same as those for NAD+. Judging by the apparent dissociation constants, negative interactions on binding the third molecule of NAD+ or NADH are more marked than those associated with the binding of the second and fourth molecules, suggesting that a major conformational change occurs at half-saturation of the tetramer with coenzyme.     

Association of cellular thiol redox status with mitogen-induced calcium mobilization and cell cycle progression in human fibroblasts

Human gingival fibroblast cultures were used to investigate the role of cellular thiol redox status in the mitogenic response. Increases in intracellular Ca2+ and cell cycle progression beyond G1 were followed as parameters of cellular mitogen-induced responses. Ethionine provided a G1 stage synchronization and altered the cellular redox poise as measured by the ratio NAD(P)H/NAD(P)+. Cultures harvested immediately after the 6 day ethionine low-serum synchronization showed a significant oxidation of their redox poise. Synchronized cultures, which were also glutathione (GSH) depleted, still showed an oxidized redox poise and significantly reduced GSH levels following a 24 hr incubation in drug-free, rich medium. Cellular reduced nicotinamide nucleotide levels correlated strongly (r = 0.995) with capacity to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). The sustained mitogenic response, as determined by cell cycle progression beyond G1, was also found to be interrelated with the cellular thiol redox status. Following a 24 hr recovery incubation in serum-rich medium, formerly synchronized cultures showed a rebound of their redox poise to a more reduced state and significant cell cycle progression beyond G1. In contrast, synchronized, GSH-depleted cultures did not progress and showed population distributions similar to those of cultures harvested immediately postsynchronization. Upon recovery of cellular GSH and reduced nicotinamide nucleotide levels, formerly GSH-depleted, growth-arrested cultures resumed cell cycle progression. The results suggest that the cellular response to specific mitogens is interrelated with the cellular thiol redox status. Cells that possess a thiol redox status below a threshold response point may have compromised Ca2+ sequestration and/or mobilization and therefore may be incapable of initiating the mitogen induced response cascade that culminates in cell cycle progression.     

Reaction of complex I of the mitochondrial electron transport chain with artificial oxidizers

The steady-state kinetics of oxidation of the mitochondrial NADH: ubiquinone oxidoreductase (complex I, EC 1.6.99.3) by artificial electron acceptors--p-quinones and inorganic complexes has been investigated. A limiting stage in the NADH: ferricyanide reductase reaction is a reductive half-reaction. Ferricyanide interacts with negative-charged protein groups taking part in the NADH binding. The rate constants of the quinone reduction by complex I vary from 1.10(6) to 4.10(3) M-1s-1. The NADH, NAD+ and ADP-ribose inhibition data indicate that oxidizers in the rotenono-insensitive reaction interact with the redox centre near the NAD+/NADH binding site, most probably with FMN.     

The coenzyme-binding characteristics of highly purified preparations of sheep liver cytoplasmic aldehyde dehydrogenase.

The binding of NADH and NAD+ by cytoplasmic aldehyde dehydrogenase was studied by various direct and indirect methods. At pH 7.0 at 25 degrees C there appears to be approx. 1 binding site for both nucleotides per 200 000 daltons of protein, although the NAD+-binding results are rather uncertain. Estimates of the dissociation constants of the E . NADH and E . NAD+ complexes under the stated conditions are also presented. Preparations of enzyme are sometimes found to contain significant amounts of very tightly bound NAD+ and NADH. The implications of these findings are discussed.     

Parameters affecting the chemical work output of a hybrid photoelectrochemical biofuel cell.

A hybrid photoelectrochemical biofuel cell employing the photoanode architecture of a dye-sensitized solar cell has been assembled. A porphyrin dye sensitizes a TiO(2) semiconductor over the visible range to beyond 650 nm. Photoinduced charge separation at the dye-TiO(2) interface results in electron migration to a cathode, and the holes generated on surface bound dyes oxidize soluble electron mediators. The increased [Ox] : [Red] ratio of the mediator drives the solution-based enzymatic oxidation of appropriate substrates. In this report we investigate how the accumulation of anodic and cathodic products limits cell performance. The NAD(+)/NADH and benzoquinone/hydroquinone redox couples were studied as sacrificial electron donors in the absence of appropriate enzymes or substrates. Comparatively poor cell performance was observed using the benzoquinone/hydroquinone couple. This effect is explained in terms of rapid charge recombination by electron donation from the electrode to benzoquinone in solution, as compared to much less recombination with NAD(+). With the NAD(+)/NADH couple the cell performance is relatively independent of the redox poise of the anode solution, but limited by accumulation of reduction products in the cathodic compartment. Using the NAD(+)/NADH couple, the photochemical reforming of ethanol to hydrogen was demonstrated under conditions where the process would be endergonic in the dark.     

Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function

Respiration in cyanobacterial thylakoid membranes is interwoven with photosynthetic processes. We have constructed a range of mutants that are impaired in several combinations of respiratory and photosynthetic electron transport complexes and have examined the relative effects on the redox state of the plastoquinone (PQ) pool by using a quinone electrode. Succinate dehydrogenase has a major effect on the PQ redox poise, as mutants lacking this enzyme showed a much more oxidized PQ pool. Mutants lacking type I and II NAD(P)H dehydrogenases also had more oxidized PQ pools. However, in the mutant lacking type I NADPH dehydrogenase, succinate was essentially absent and effective respiratory electron donation to the PQ pool could be established after addition of 1 mM succinate. Therefore, lack of the type I NADPH dehydrogenase had an indirect effect on the PQ pool redox state. The electron donation capacity of succinate dehydrogenase was found to be an order of magnitude larger than that of type I and II NAD(P)H dehydrogenases. The reason for the oxidized PQ pool upon inactivation of type II NADH dehydrogenase may be related to the facts that the NAD pool in the cell is much smaller than that of NADP and that the NAD pool is fully reduced in the mutant without type II NADH dehydrogenase, thus causing regulatory inhibition. The results indicate that succinate dehydrogenase is the main respiratory electron transfer pathway into the PQ pool and that type I and II NAD(P)H dehydrogenases regulate the reduction level of NADP and NAD, which, in turn, affects respiratory electron flow through succinate dehydrogenase.     

胞内氧化还原水平对嗜热厌氧乙醇菌 发酵代谢的影响

辅酶NADH/NAD+在细胞内氧化还原反应中起着重要的作用,是细胞生长和能量代谢必不可少的辅因子。调节微生物胞内NADH/NAD+的比率是定向改变微生物代谢,高效获得目标代谢产物的有效手段。嗜热厌氧乙醇菌(Thermoanaerobacter ethanolicus)是高温厌氧菌中乙醇产量较高的代表性菌株,本文利用不同氧化还原态的碳源改变T.ethanolicus的胞内NADH/NAD+含量和比例,进而研究了其对细胞生长、代谢产物分布的影响。以不同比例的葡萄糖/甘露醇作为混合碳源发酵,胞内氧化还原水平、细胞的生长特性、代谢产物都发生了不同程度的差异,以葡萄糖作为唯一碳源进行培养时,T.ethanolicus生长良好,乙醇产量为0.79g/L,但胞内NADH/NAD+比值和乙醇/乙酸的比值都比较低,分别为0.47和4.82;随着葡萄糖在混合碳源中比例的下降,NADH/NAD+比值增高,发酵产物中乙醇/乙酸比值也呈现上升的趋势。而以甘露醇作为唯一碳源时,发酵产物中乙醇浓度为0.389g/L,NADH/NAD+比值和乙醇/乙酸的比值分别为1.04和16.0。     

Specific interactions of 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase with coenzymes.

The binding of oxidized and reduced coenzyme (NAD+ and NADH) to 3-phosphoglyceroyl-glyceraldehyde-3-phosphate dehydrogenase has been studied spectrophotometrically and fluorimetrically. The binding of NAD+ to the acylated sturgeon enzyme is characterized by a significant quenching of the enzyme fluorescence (about 25%) and the induction of a difference spectrum in the ultraviolet absorbance region of the enzyme. Both of these spectroscopic properties are quantitatively distinguishable from those of the corresponding binary enzyme-NAD+ complex. Binding isotherms estimated by gel filtration of the acylated enzyme are in close agreement to those obtained by spectrophotometric and fluorimetric titrations. Up to four NAD+ molecules are bound to the enzyme tetramer. No anticooperativity can be detected in the binding of oxidized coenzyme, which is well described on the basis of a single class of four binding sites with a dissociation constant of 25 muM at 10 degrees C, pH 7.0. The binding of NADH to the acylenzyme has been characterized spectrophotometrically. The absorption band of the dihydronicotinamide moiety of the coenzyme is blue-shifted to 335 nm with respect to free NADH. In addition, a large hypochromicity (23%) is observed together with a significant increase of the bandwidth at half height of this absorption band. This last property is specific to the acylenzyme-DADH complex, since it disappears upon arsenolysis of the acylenzyme. The binding affinity of NADH to the acylated enzyme has been estimated by performing simultaneous spectrophotometric and fluorimetric titrations of the NADH appearance upon addition of NAD+ to a mixture of enzyme and excess glyceraldehyde 3-phosphate. In contrast to NAD+, the reduced coenzyme NADH appears to be relatively strongly bound to the acylated enzyme, the dissociation constant of the acylenzyme-NADH complex being estimated as 2.0 muM at 25 degrees C. In addition a large quenching of the NADH fluorescence (about 83%) is observed. The comparison of the dissociation constants of the coenzyme-acylenzyme complexes and the corresponding Michaelis constants suggests a reaction mechanism of the enzyme in which significant formation and dissociation of NAD+-acylenzyme and NADH-acylenzyme complexes occur. Under physiological conditions the activity of the enzyme can be regulated by the ratio of oxidized and reduced coenzymes. Possible reasons for the lack of anticooperativity in coenzyme binding to the acylated form of the enzyme are discussed.