Cloning and characterization of a thermostable H2O-forming NADH oxidase from Lactobacillus rhamnosus

NADH oxidase (Nox) catalyzes the conversion of NADH to NAD(+). A previously uncharacterized Nox gene (LrNox) was cloned from Lactobacillus rhamnosus and overexpressed in Escherichia coli BL21(DE3). Sequence analysis revealed an open reading frame of 1359 bp, capable of encoding a polypeptide of 453 amino acid residues. The molecular mass of the purified LrNox enzyme was estimated to be ~50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 100 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme had optimal activity at pH 5.6 and temperature 65 °C, and k(cat)/K(m) of 3.77×10(7) s(-1) M(-1), the highest ever reported. Heat inactivation studies revealed that LrNox had high thermostability, with a half-life of 120 min at 80 °C. Molecular dynamics simulation studies shed light on the factors contributing to the high activity of LrNox. Although the properties of Nox from several microorganisms have been reported, this is the first report on the characterization of a recombinant H(2)O-forming Nox with high activity and thermostability. The characteristics of the LrNox enzyme could prove to be of interest in industrial applications such as NAD(+) regeneration.     

Characterization of H2O-forming NADH oxidase from Streptococcus pyogenes and its application in l-rare sugar production

A nicotinamide adenine dinucleotide (NADH) oxidase from Streptococcus pyogenes MGAS10394 (SpNox) was cloned and overexpressed in Escherichia coli BL21 (DE3). The purified SpNox enzyme had optimal pH and temperature of 7.0 and 55°C, respectively, with a K(m) of 27.0μM and a k(cat)/K(m) of 1.1×10(7)s(-1)M(-1). SpNox showed the highest activity among all known NADH oxidases, and site-directed mutagenesis and docking analysis shed light on the molecular basis of its unusually high activity. The characteristics of SpNox may prove to be useful for NAD(+) regeneration in the production of l-rare sugar.     

Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, reduction of NAD(+) to NADH occurs in dissimilatory as well as in assimilatory reactions. This review discusses mechanisms for reoxidation of NADH in this yeast, with special emphasis on the metabolic compartmentation that occurs as a consequence of the impermeability of the mitochondrial inner membrane for NADH and NAD(+). At least five mechanisms of NADH reoxidation exist in S. cerevisiae. These are: (1) alcoholic fermentation; (2) glycerol production; (3) respiration of cytosolic NADH via external mitochondrial NADH dehydrogenases; (4) respiration of cytosolic NADH via the glycerol-3-phosphate shuttle; and (5) oxidation of intramitochondrial NADH via a mitochondrial 'internal' NADH dehydrogenase. Furthermore, in vivo evidence indicates that NADH redox equivalents can be shuttled across the mitochondrial inner membrane by an ethanol-acetaldehyde shuttle. Several other redox-shuttle mechanisms might occur in S. cerevisiae, including a malate-oxaloacetate shuttle, a malate-aspartate shuttle and a malate-pyruvate shuttle. Although key enzymes and transporters for these shuttles are present, there is as yet no consistent evidence for their in vivo activity. Activity of several other shuttles, including the malate-citrate and fatty acid shuttles, can be ruled out based on the absence of key enzymes or transporters. Quantitative physiological analysis of defined mutants has been important in identifying several parallel pathways for reoxidation of cytosolic and intramitochondrial NADH. The major challenge that lies ahead is to elucidate the physiological function of parallel pathways for NADH oxidation in wild-type cells, both under steady-state and transient-state conditions. This requires the development of techniques for accurate measurement of intracellular metabolite concentrations in separate metabolic compartments.     

利用辅因子工程策略提高酿酒酵母中S-腺苷蛋氨酸的生物合成

通过代谢工程策略改造酿酒酵母胞内辅因子的形式和浓度,分析辅因子NADPH对于产物S-腺苷蛋氨酸(SAM)合成的作用并总结能量代谢和其他物质代谢的规律,为高产SAM菌株的代谢工程改造提供理论基础。由于酿酒酵母中的NADPH在线粒体和细胞质中的代谢相对独立,因此以酿酒酵母BY4741单倍体模式菌株为研究对象,研究了不同亚细胞结构内NADPH对于产物合成的影响。通过激光共聚焦显微镜证实了NADH激酶在酿酒酵母线粒体和细胞质中的表达。实验结果表明NADPH的提高有利于酿酒酵母胞内SAM的合成。发酵24 h,菌株NBYSM-1胞内SAM浓度较对照菌提高3.28倍,菌株NBYSM-2胞内SAM浓度提高1.79倍。其中重组菌株NBYSM-1合成SAM的能力和胞内NADPH/NADP~+比率均明显高于重组菌株NBYSM-2。因此,NADPH调控策略有望成为提高SAM产量的有力工具并应用于其他辅因子依赖化合物的合成。     

过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸

[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.     

Physiological and genetic engineering of cytosolic redox metabolism in Saccharomyces cerevisiae for improved glycerol production

Previous metabolic engineering strategies for improving glycerol production by Saccharomyces cerevisiae were constrained to a maximum theoretical glycerol yield of 1 mol.(molglucose)(-1) due to the introduction of rigid carbon, ATP or redox stoichiometries. In the present study, we sought to circumvent these constraints by (i) maintaining flexibility at fructose-1,6-bisphosphatase and triosephosphate isomerase, while (ii) eliminating reactions that compete with glycerol formation for cytosolic NADH and (iii) enabling oxidative catabolism within the mitochondrial matrix. In aerobic, glucose-grown batch cultures a S. cerevisiae strain, in which the pyruvate decarboxylases the external NADH dehydrogenases and the respiratory chain-linked glycerol-3-phosphate dehydrogenase were deleted for this purpose, produced glycerol at a yield of 0.90 mol.(molglucose)(-1). In aerobic glucose-limited chemostat cultures, the glycerol yield was ca. 25% lower, suggesting the involvement of an alternative glucose-sensitive mechanism for oxidation of cytosolic NADH. Nevertheless, in vivo generation of additional cytosolic NADH by co-feeding of formate to aerobic, glucose-limited chemostat cultures increased the glycerol yield on glucose to 1.08 mol mol(-1). To our knowledge, this is the highest glycerol yield reported for S. cerevisiae.     

Organization and regulation of the cytosolic NADH metabolism in the yeast Saccharomyces cerevisiae

Keeping a cytosolic redox balance is a prerequisite for living cells in order to maintain a metabolic activity and enable growth. During growth of Saccharomyces cerevisiae, an excess of NADH is generated in the cytosol. Aerobically, it has been shown that the external NADH dehydrogenase, Nde1p and Nde2p, as well as the glycerol-3-phosphate dehydrogenase shuttle, comprising the cytoplasmic glycerol-3-phosphate dehydrogenase, Gpdlp, and the mitochondrial glycerol-3-phosphate dehydrogenase, Gut2p, are the most important mechanisms for mitochondrial oxidation of cytosolic NADH. In this review we summarize the recent results showing (i) the contribution of each of the mechanisms involved in mitochondrial oxidation of the cytosolic NADH, under different physiological situations; (ii) the kinetic and structural properties of these metabolic pathways in order to channel NADH from cytosolic dehydrogenases to the inner mitochondrial membrane and (iii) the organization in supramolecular complexes and, the peculiar ensuing kinetic regulation of some of the enzymes (i.e. Gut2p inhibition by external NADH dehydrogenase activity) leading to a highly integrated functioning of enzymes having a similar physiological function. The cell physiological consequences of such an organized and regulated network are discussed.     

Expression of spinach glycolate oxidase in Saccharomyces cerevisiae: purification and characterization.

Glycolate oxidase from spinach has been expressed in Saccharomyces cerevisiae. The active enzyme was purified to near-homogeneity (purification factor approximately 1400-fold) by means of hydroxyapatite and anion-exchange chromatography. The purified glycolate oxidase is nonfluorescent and has absorbance peaks at 448 (epsilon = 9200 M-1 cm-1) and 346 nm in 0.1 M phosphate buffer, pH 8.3. The large bathochromic shift of the near-UV band indicates that the N(3) position is deprotonated at pH 8.3. A pH titration revealed that the pK of the N(3) is shifted from 10.3 in free flavin to 6.4 in glycolate oxidase. Glycolate oxidase is competitively inhibited by oxalate with a Kd of 0.24 mM at 4 degrees C in 0.1 M phosphate buffer, pH 8.3. Three pieces of evidence demonstrate that glycolate oxidase stabilizes a negative charge at the N(1)-C(2 = O) locus: the enzyme forms a tight sulfite complex with a Kd of 2.7 x 10(-7) M and stabilizes the anionic flavosemiquinone and the benzoquinoid form of 8-mercapto-FMN. Steady-state analysis at pH 8.3, 4 degrees C, yielded a Km = 1 x 10(-3) M for glycolate and Km = 2.1 x 10(-4) M for oxygen. The turnover number has been determined to be 20 s-1. Stopped-flow studies of the reductive (k = 25 s-1) and oxidative (k = 8.5 x 10(4) M-1 s-1) half-reactions have identified the reduction of glycolate oxidase to be the rate-limiting step.     

Decreased xylitol formation during xylose fermentation in Saccharomyces cerevisiae due to overexpression of water-forming NADH oxidase

The recombinant xylose-fermenting Saccharomyces cerevisiae strain harboring xylose reductase (XR) and xylitol dehydrogenase (XDH) from Scheffersomyces stipitis requires NADPH and NAD(+), creates cofactor imbalance, and causes xylitol accumulation during growth on d-xylose. To solve this problem, noxE, encoding a water-forming NADH oxidase from Lactococcus lactis driven by the PGK1 promoter, was introduced into the xylose-utilizing yeast strain KAM-3X. A cofactor microcycle was set up between the utilization of NAD(+) by XDH and the formation of NAD(+) by water-forming NADH oxidase. Overexpression of noxE significantly decreased xylitol formation and increased final ethanol production during xylose fermentation. Under xylose fermentation conditions with an initial d-xylose concentration of 50 g/liter, the xylitol yields for of KAM-3X(pPGK1-noxE) and control strain KAM-3X were 0.058 g/g xylose and 0.191 g/g, respectively, which showed a 69.63% decrease owing to noxE overexpression; the ethanol yields were 0.294 g/g for KAM-3X(pPGK1-noxE) and 0.211 g/g for the control strain KAM-3X, which indicated a 39.33% increase due to noxE overexpression. At the same time, the glycerol yield also was reduced by 53.85% on account of the decrease in the NADH pool caused by overexpression of noxE.     

Comparison of glycolytic NADH oscillations in yeasts Saccharomyces cerevisiae and Saccharomyces carlsbergensis

The possible mechanism of synchronization of NADH oscillations in yeasts were studied. It was shown that the synchronization time depends on cell concentration in suspension. Synchronization of oscillations after acetaldehyde addition was found in Saccharomyces carlsbergensis whereas in S. cerevisiae oscillations were synchronized after adding potassium cyanide. It is possible, that synchronization of oscillations in S. cerevisiae requires low concentration of acetaldehyde and the high acetaldehyde concentration synchronizes oscillations in S. carlsbergensis. In addition, a possible mechanism of synchronization by acetaldehyde in proposed.     

Metabolic Impact of Increased NADH Availability in Saccharomyces cerevisiae

Engineering the level of metabolic cofactors to manipulate metabolic flux is emerging as an attractive strategy for bioprocess applications. We present the metabolic consequences of increasing NADH in the cytosol and the mitochondria of Saccharomyces cerevisiae. In a strain that was disabled in formate metabolism, we either overexpressed the native NAD⁺-dependent formate dehydrogenase in the cytosol or directed it into the mitochondria by fusing it with the mitochondrial signal sequence encoded by the CYB2 gene. Upon exposure to formate, the mutant strains readily consumed formate and induced fermentative metabolism even under conditions of glucose derepression. Cytosolic overexpression of formate dehydrogenase resulted in the production of glycerol, while when this enzyme was directed into the mitochondria, we observed glycerol and ethanol production. Clearly, these results point toward different patterns of compartmental regulation of redox homeostasis. When pulsed with formate, S. cerevisiae cells growing in a steady state on glucose immediately consumed formate. However, formate consumption ceased after 20 min. Our analysis revealed that metabolites at key branch points of metabolic pathways were affected the most by the genetic perturbations and that the intracellular concentrations of sugar phosphates were specifically affected by time. In conclusion, the results have implications for the design of metabolic networks in yeast for industrial applications.The traditional use of baker''s yeast, Saccharomyces cerevisiae, for ethanol production has resulted in the accumulation of substantial information about its genetics, metabolism, and process development. Consequently, the collection of compounds that are produced using S. cerevisiae has expanded to include organic acids and even secondary metabolites (1, 25, 28). Unlike ethanol, many of these products are not redox neutral relative to commonly used substrates such as glucose. Therefore, in addition to stoichiometry, redox constraints play an important role in the formation of the products. Additional reducing power has to be supplied to produce compounds whose degree of reduction is higher than that of the substrate. On the other hand, producing compounds with a degree of reduction lower than that of the substrate will force the synthesis of other compounds with higher degrees of reduction to compensate for excess reducing power generated from substrate oxidation. These constraints may decrease the product yield substantially.The catabolic currency that balances the degree of reduction between the substrate and the products is usually NADH. In S. cerevisiae, NADH is produced in the cytosol by mainly glyceraldehyde-3-phosphate dehydrogenase and other assimilatory reaction enzymes (35). In the mitochondria, NADH is formed in the tricarboxylic acid (TCA) cycle and the reaction of the pyruvate dehydrogenase complex. Cytosolic NADH is oxidized by the glycerol-3-phosphate shuttle or the external cytosolic NADH dehydrogenases, which are part of the electron transport chain (21). NADH can be transported across the outer mitochondrial membrane (18, 19) but not across the inner mitochondrial membrane (39). Therefore, a dedicated internal mitochondrial NADH dehydrogenase is required to oxidize mitochondrial NADH as part of the electron transport chain (22). The compartmental restriction of NADH oxidation has important ramifications for metabolism and electron transport. The electrons originating from cytosolic NADH are preferred over those originating from mitochondrial NADH (6) for entrance into the electron transport chain. The direct consequence of preferential utilization of cytosolic NADH is a higher redox potential (NADH/NAD⁺) in the mitochondria than in the cytosol. Consequently, during rapid NADH synthesis, as during exponential growth, the TCA cycle ceases to operate as a cycle and branches into oxidative and reducing pathways (12).Metabolic consequences of the compartmentalization of NADH homeostasis were evident from the difference in the product formation profile upon lowering of cytosolic or mitochondrial NADH. Lowering cytosolic NADH by overexpressing bacterial NADH oxidase lowered the production of glycerol and biomass by S. cerevisiae (14, 36). On the other hand, decreasing the mitochondrial NADH level decreased ethanol production and increased the biomass yield (36). These results are likely to be a combination of effects from alleviating the feedback inhibition of the TCA cycle by mitochondrial NADH and increasing respiratory capacity due to improved efficiency of oxidative phosphorylation, as quantified by the P/O ratio (15). There are no reports that describe the effect of increasing NADH in S. cerevisiae, although formate has been used previously as a source of additional reducing power in S. cerevisiae (2, 4, 11, 23, 24, 27). Formate (HCOO⁻) is efficiently oxidized to CO₂ by NAD⁺-dependent formate dehydrogenase (27) and, therefore, cannot be used as a carbon source for biomass synthesis. Thus, using formate as an auxiliary substrate for the generation of NADH to study the effect of increased NADH may be a feasible option. Given the compartment-dependent regulation of NADH homeostasis in S. cerevisiae (36), increasing the NADH level in the cytosol is likely to elicit a response different from that obtained by increasing the NADH level in the mitochondria.The aim of the present study is to differentiate between the metabolic consequences of increasing NADH in the cytosol and those of increasing NADH in the mitochondria of S. cerevisiae. Toward this aim, we either overexpressed the native Fdh1 (NAD⁺-dependent formate dehydrogenase) in the cytosol or directed it into the mitochondria in a strain background that is otherwise devoid of formate metabolism. We present our understanding of the physiological characteristics of the mutant strains under steady-state or dynamic conditions in the presence of different levels of formate.     

Effect of electrochemical redox reaction on growth and metabolism of Saccharomyces cerevisiae as an environmental factor

The effect of an electrochemically generated oxidation-reduction potential and electric pulse on ethanol production and growth of Saccharomyces cerevisiae ATCC 26603 was experimented and compared with effects of electron mediators (neutral red, benzyl viologen, and thionine), chemical oxidants (hydrogen peroxide and hypochlorite), chemical reductants (sulfite and nitrite), oxygen, and hydrogen. The oxidation (anodic) and reduction (cathodic) potential and electric pulse activated ethanol production and growth, and changed the total soluble protein pattern of the test strain. Neutral red electrochemically reduced activated ethanol production and growth of the test strain, but benzyl viologen and thionine did not. Nitrite inhibited ethanol production but did not influence growth of the test strain. Hydrogen peroxide, hypochlorite, and sulfite did not influence ethanol production and growth of the test strain. Hydrogen and oxygen also did not influence the growth and ethanol production. It shows that the test strain may perceive electrochemically generated oxidation-reduction potential and electric pulse as an environmental factor.     

External alternative NADH dehydrogenase of Saccharomyces cerevisiae: a potential source of superoxide

Three rotenone-insensitive NADH dehydrogenases are present in the mitochondria of yeast Saccharomyces cerevisiae, which lack complex I. To elucidate the functions of these enzymes, superoxide production was determined in yeast mitochondria. The low levels of hydrogen peroxide (0.10 to 0.18 nmol/min/mg) produced in mitochondria incubated with succinate, malate, or NADH were stimulated 9-fold by antimycin A. Myxothiazol and stigmatellin blocked completely hydrogen peroxide formation with succinate or malate, indicating that the cytochrome bc(1) complex is the source of superoxide; however, these inhibitors only inhibited 46% hydrogen peroxide formation with NADH as substrate. Diphenyliodonium inhibited hydrogen peroxide formation (with NADH as substrate) by 64%. Superoxide formation, determined by EPR and acetylated cytochrome c reduction in mitochondria was stimulated by antimycin A, and partially inhibited by myxothiazol and stigmatellin. Proteinase K digestion of mitoplasts reduced 95% NADH dehydrogenase activity with a similar inhibition of superoxide production. Mild detergent treatment of the proteinase-treated mitoplasts resulted in an increase in NADH dehydrogenase activity due to the oxidation of exogenous NADH by the internal NADH dehydrogenase; however, little increase in superoxide production was observed. These results suggest that the external NADH dehydrogenase is a potential source of superoxide in S. cerevisiae mitochondria.     

The interplay between sulphur and selenium metabolism influences the intracellular redox balance in Saccharomyces cerevisiae

Selenium (Se) is an essential element for most eukaryotic organisms, including humans. The balance between Se toxicity and its beneficial effects is very delicate. It has been demonstrated that a diet enriched with Se has cancer prevention potential in humans. The most popular commercial Se supplementation is selenized yeast, which is produced in a fermentation process using an inorganic source of Se. Here, we show that the uptake of Se, Se toxic effects and intracellular Se-metabolite profile are largely influenced by the level of sulphur source supplied during the fermentation. A Yap1-dependent oxidative stress response is active when yeast actively metabolizes Se, and this response is linked to the generation of intracellular redox imbalance. The redox imbalance derives from a disproportionate ratio between the reduced and oxidized forms of glutathione and also from the influence of Se metabolism on the central carbon metabolism. The observed increase in glycerol production rate, concomitant with the inhibition of ethanol formation in the presence of Se, can be ascribed to the occurrence of redox imbalance that triggers glycerol biosynthesis to replenish the pool of NAD(+) .     

Identification of a Cytosolically Directed NADH Dehydrogenase in Mitochondria of Saccharomyces cerevisiae

The reoxidation of NADH generated in reactions within the mitochondrial matrix of Saccharomyces cerevisiae is catalyzed by an NADH dehydrogenase designated Ndi1p (C. A. M. Marres, S. de Vries, and L. A. Grivell, Eur. J. Biochem. 195:857–862, 1991). Gene disruption analysis was used to examine possible metabolic functions of two proteins encoded by open reading frames having significant primary sequence similarity to Ndi1p. Disruption of the gene designated NDH1 results in a threefold reduction in total mitochondrial NADH dehydrogenase activity in cells cultivated with glucose and in a fourfold reduction in the respiration of isolated mitochondria with NADH as the substrate. Thus, Ndh1p appears to be a mitochondrial dehydrogenase capable of using exogenous NADH. Disruption of a closely related gene designated NDH2 has no effect on these properties. Growth phenotype analyses suggest that the external NADH dehydrogenase activity of Ndh1p is important for optimum cellular growth with a number of nonfermentable carbon sources, including ethanol. Codisruption of NDH1 and genes encoding malate dehydrogenases essentially eliminates growth on nonfermentable carbon sources, suggesting that the external mitochondrial NADH dehydrogenase and the malate-aspartate shuttle may both contribute to reoxidation of cytosolic NADH under these growth conditions.     

Cofactor assisted gating mechanism in the active site of NADH oxidase from Thermus thermophilus

NADH oxidase (NOX) from Thermus thermophilus is a member of a structurally homologous flavoprotein family of nitroreductases and flavin reductases. The importance of local conformational dynamics in the active site of NOX has been recently demonstrated. The enzyme activity was increased by 250% in the presence of 1 M urea with no apparent perturbation of the native structure of the protein. The present in silico results correlate with the in vitro data and suggest the possible explanation about the effect of urea on NOX activity at the molecular level. Both, X-ray structure and molecular dynamics (MD) simulations, show open conformation of the active site represented by approximately 0.9 nm distance between the indole ring of Trp47 and the isoalloxazine ring of FMN412. In this conformation, the substrate molecule can bind in the active site without sterical restraints. MD simulations also indicate more stable conformation of the active site called "closed" conformation. In this conformation, Trp47 and the isoalloxazine ring of FMN412 are so close to each other (approximately 0.5 nm) that the substrate molecule is unable to bind between them without perturbing this conformation. The open/close transition of the active site between Trp47 and the flavin ring is accompanied by release of the "tightly" bound water molecule from the active site--cofactor assisted gating mechanism. The presence of urea in aqueous solutions of NOX prohibits closing of the active site and even unlocks the closed active site because of the concomitant binding of a urea molecule in the active site cavity. The binding of urea in the active site is stabilized by formation of one/two persistent hydrogen bonds involving the carbonyl group of the urea molecule. Our report represents the first MD study of an enzyme from the novel flavoprotein family of nitroreductases and flavin reductases. The common occurrence of aromatic residues covering the active sites in homologous enzymes suggests the possibility of a general gating mechanism and the importance of local dynamics within this flavoprotein family.     

辅酶工程在酿酒酵母木糖代谢工程中的研究进展

辅酶工程（cofactor engineering）是代谢工程的一个重要分支,它通过改变辅酶的再生途径,达到改变细胞内代谢产物构成的目的。介绍了酿酒酵母(Saccharomyces cerevisiae)木糖代谢工程中,利用辅酶工程解决氧化还原平衡问题的研究进展,包括引入转氢酶系统,增加代谢中可利用的NADPH,实现NADH的厌氧氧化等策略。同时介绍了改变XR、XDH辅酶偏好的研究进展。