Bioenergetic Alterations of Metabolic Redox Coenzymes as NADH,FAD and FMN by Means of Fluorescence Lifetime Imaging Techniques

Metabolic FLIM (fluorescence lifetime imaging) is used to image bioenergetic status in cells and tissue. Whereas an attribution of the fluorescence lifetime of coenzymes as an indicator for cell metabolism is mainly accepted, it is debated whether this is valid for the redox state of cells. In this regard, an innovative algorithm using the lifetime characteristics of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) to calculate the fluorescence lifetime induced redox ratio (FLIRR) has been reported so far. We extended the FLIRR approach and present new results, which includes FLIM data of the various enzymes, such as NAD(P)H, FAD, as well as flavin mononucleotide (FMN). Our algorithm uses a two-exponential fitting procedure for the NAD(P)H autofluorescence and a three-exponential fit of the flavin signal. By extending the FLIRR approach, we introduced FLIRR1 as protein-bound NAD(P)H related to protein-bound FAD, FLIRR2 as protein-bound NAD(P)H related to free (unbound) FAD and FLIRR3 as protein-bound NAD(P)H related to protein-bound FMN. We compared the significance of extended FLIRR to the metabolic index, defined as the ratio of protein-bound NAD(P)H to free NAD(P)H. The statistically significant difference for tumor and normal cells was found to be highest for FLIRR1.     

Characterization of a Novel Thermostable Dye-Linked l-Lactate Dehydrogenase Complex and Its Application in Electrochemical Detection

Flavoenzyme dye-linked l-lactate dehydrogenase (Dye-LDH) is primarily involved in energy generation through electron transfer and exhibits potential utility in electrochemical devices. In this study, a gene encoding a Dye-LDH homolog was identified in a hyperthermophilic archaeon, Sulfurisphaera tokodaii. This gene was part of an operon that consisted of four genes that were tandemly arranged in the Sf. tokodaii genome in the following order: stk_16540, stk_16550 (dye-ldh homolog), stk_16560, and stk_16570. This gene cluster was expressed in an archaeal host, Sulfolobus acidocaldarius, and the produced enzyme was purified to homogeneity and characterized. The purified recombinant enzyme exhibited Dye-LDH activity and consisted of two different subunits (products of stk_16540 (α) and stk_16550 (β)), forming a heterohexameric structure (α3β3) with a molecular mass of approximately 253 kDa. Dye-LDH also exhibited excellent stability, retaining full activity upon incubation at 70 °C for 10 min and up to 80% activity after 30 min at 50 °C and pH 6.5–8.0. A quasi-direct electron transfer (DET)-type Dye-LDH was successfully developed by modification of the recombinant enzyme with an artificial redox mediator, phenazine ethosulfate, through amine groups on the enzyme’s surface. This study is the first report describing the development of a quasi-DET-type enzyme by using thermostable Dye-LDH.     

Iron Mobilization from Ferritin in Yeast Cell Lysate and Physiological Implications

Most in vitro iron mobilization studies from ferritin have been performed in aqueous buffered solutions using a variety of reducing substances. The kinetics of iron mobilization from ferritin in a medium that resembles the complex milieu of cells could dramatically differ from those in aqueous solutions, and to our knowledge, no such studies have been performed. Here, we have studied the kinetics of iron release from ferritin in fresh yeast cell lysates and examined the effect of cellular metabolites on this process. Our results show that iron release from ferritin in buffer is extremely slow compared to cell lysate under identical experimental conditions, suggesting that certain cellular metabolites present in yeast cell lysate facilitate the reductive release of ferric iron from the ferritin core. Using filtration membranes with different molecular weight cut-offs (3, 10, 30, 50, and 100 kDa), we demonstrate that a cellular component >50 kDa is implicated in the reductive release of iron. When the cell lysate was washed three times with buffer, or when NADPH was omitted from the solution, a dramatic decrease in iron mobilization rates was observed. The addition of physiological concentrations of free flavins, such as FMN, FAD, and riboflavin showed about a two-fold increase in the amount of released iron. Notably, all iron release kinetics occurred while the solution oxygen level was still high. Altogether, our results indicate that in addition to ferritin proteolysis, there exists an auxiliary iron reductive mechanism that involves long-range electron transfer reactions facilitated by the ferritin shell. The physiological implications of such iron reductive mechanisms are discussed.     

Bacterial Over-Expression and Purification of the 3′phosphoadenosine 5′phosphosulfate (PAPS) Reductase Domain of Human FAD Synthase: Functional Characterization and Homology Modeling

FAD synthase (FADS, EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor, FAD. Human FADS is organized in two domains: -the 3′phosphoadenosine 5′phosphosulfate (PAPS) reductase domain, similar to yeast Fad1p, at the C-terminus, and -the resembling molybdopterin-binding domain at the N-terminus. To understand whether the PAPS reductase domain of hFADS is sufficient to catalyze FAD synthesis, per se, and to investigate the role of the molybdopterin-binding domain, a soluble “truncated” form of hFADS lacking the N-terminal domain (Δ_1-328-hFADS) has been over-produced and purified to homogeneity as a recombinant His-tagged protein. The recombinant Δ_1-328-hFADS binds one mole of FAD product very tightly as the wild-type enzyme. Under turnover conditions, it catalyzes FAD assembly from ATP and FMN and, at a much lower rate, FAD pyrophosphorolytic hydrolysis. The Δ_1-328-hFADS enzyme shows a slight, but not significant, change of K_m values (0.24 and 6.23 μM for FMN and ATP, respectively) and of k_cat (4.2 × 10⁻² s⁻¹) compared to wild-type protein in the forward direction. These results demonstrate that the molybdopterin-binding domain is not strictly required for catalysis. Its regulatory role is discussed in light of changes in divalent cations sensitivity of the Δ_1-328-hFADS versus wild-type protein.     

Crystal Structure of Dimeric Flavodoxin from Desulfovibrio gigas Suggests a Potential Binding Region for the Electron-Transferring Partner

Flavodoxins, which exist widely in microorganisms, have been found in various pathways with multiple physiological functions. The flavodoxin (Fld) containing the cofactor flavin mononucleotide (FMN) from sulfur-reducing bacteria Desulfovibrio gigas (D. gigas) is a short-chain enzyme that comprises 146 residues with a molecular mass of 15 kDa and plays important roles in the electron-transfer chain. To investigate its structure, we purified this Fld directly from anaerobically grown D. gigas cells. The crystal structure of Fld, determined at resolution 1.3 Å, is a dimer with two FMN packing in an orientation head to head at a distance of 17 Å, which generates a long and connected negatively charged region. Two loops, Thr59–Asp63 and Asp95–Tyr100, are located in the negatively charged region and between two FMN, and are structurally dynamic. An analysis of each monomer shows that the structure of Fld is in a semiquinone state; the positions of FMN and the surrounding residues in the active site deviate. The crystal structure of Fld from D. gigas agrees with a dimeric form in the solution state. The dimerization area, dynamic characteristics and structure variations between monomers enable us to identify a possible binding area for its functional partners.     

Evidence for the presence of a FAD pyrophosphatase and a FMN phosphohydrolase in yeast mitochondria: a possible role in flavin homeostasis

Despite the crucial roles of flavin cofactors in metabolism, we know little about the enzymes responsible for the turnover of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and their subcellular localization. The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation, because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial function in both bioenergetics and cellular regulation. In this context, the capability of yeast mitochondria to metabolize externally added and endogenous FAD and FMN was investigated and use was made of purified and bioenergetically active mitochondria prepared starting from the Saccharomyces cerevisiae cell. To determine whether flavin metabolism can occur, the amounts of flavins in aliquots of neutralized perchloric extracts of both spheroplasts and mitochondria were measured by HPLC, and the competence of S. cerevisiae mitochondria to metabolize FAD and FMN was investigated both spectroscopically and via HPLC. FAD deadenylation and FMN dephosphorylation were studied with respect to dependence on substrate concentration, pH profile and inhibitor sensitivity. The existence of two novel mitochondrial FAD pyrophosphatase (diphosphatase) (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) activities, which catalyse the reactions FAD + H₂O → FMN + AMP and FMN + H₂O → riboflavin + Pi respectively, is here shown by fractionation studies. Considering cytosolic riboflavin, FMN and FAD concentrations, as calculated by measuring both spheroplast and mitochondrial contents via HPLC, probably mitochondria play a major role in regulating the flavin pool in yeast and in relation to flavin homeostasis. Copyright © 2011 John Wiley & Sons, Ltd.     

Role of Key Residues at the Flavin Mononucleotide (FMN):Adenylyltransferase Catalytic Site of the Bifunctional Riboflavin Kinase/Flavin Adenine Dinucleotide (FAD) Synthetase from Corynebacterium ammoniagenes

In mammals and in yeast the conversion of Riboflavin (RF) into flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) is catalysed by the sequential action of two enzymes: an ATP:riboflavin kinase (RFK) and an ATP:FMN adenylyltransferase (FMNAT). However, most prokaryotes depend on a single bifunctional enzyme, FAD synthetase (FADS), which folds into two modules: the C-terminal associated with RFK activity and the N-terminal associated with FMNAT activity. Sequence and structural analysis suggest that the 28-HxGH-31, 123-Gx(D/N)-125 and 161-xxSSTxxR-168 motifs from FADS must be involved in ATP stabilisation for the adenylylation of FMN, as well as in FAD stabilisation for FAD phyrophosphorolysis. Mutants were produced at these motifs in the Corynebacterium ammoniagenes FADS (CaFADS). Their effects on the kinetic parameters of CaFADS activities (RFK, FMNAT and FAD pyrophosphorilase), and on substrates and product binding properties indicate that H28, H31, N125 and S164 contribute to the geometry of the catalytically competent complexes at the FMNAT-module of CaFADS.     

大肠杆菌中核黄素的生物合成途径改造及生产

利用PCR技术从枯草芽孢杆菌(Bacillus subtilis 168)中扩增出3.5 kb的核黄素操纵子,将其分别连接到不同拷贝数的表达载体pSC101、p15A、pBR322,得到重组载体pSC101-BSrib、p15A-BSrib和pBR322-BSrib,并分别转化到大肠杆菌(Escherichia coli K-12 MG1655)。对含有核黄素操纵子的重组大肠杆菌进行摇瓶发酵,结果表明其核黄素合成能力随着质粒拷贝数的增加而增强。随后对E. coli K-12 MG1655 ECX3菌株的诱导剂IPTG浓度和发酵温度进行了优化。结果显示,0.1 mmol·L^-1 IPTG和42 ℃为核黄素生产的最适宜条件。在此条件下,菌株ECX3在LB培养基中核黄素产量达到251.4 mg·L^-1。最后,通过无痕基因操作技术,减弱了工程菌株ECX3核黄素激酶/黄素腺嘌呤二核苷酸氨酰转移酶(ribF)的表达以减少核黄素转化为FMN和FAD,摇瓶中工程菌株ECX4的核黄素产量提高到292.3 mg·L^-1。     

快速监控FMN在热连轧上的应用研究

研究了快速监控FMN在热连轧上的应用,提高头部命中率是投入绝对AGC所要求的,它的重要手段是靠提高设定模型精度、模型自学习的收敛速度。为进一步提高带坯全长厚控精度及控制品质,依靠液压压下快速移动特性及利用快速监控FMN功能。以热连轧模型增量方程为核心,对不同轧制规程,给出了兼顾板厚与板形FMN动作表。在鞍钢1700ASP热轧现场中的实际应用表明,此算法是正确的,它可大幅度地改善板卷头部的厚度精度。