Structures of iron-dependent alcohol dehydrogenase 2 from Zymomonas mobilis ZM4 with and without NAD+ cofactor

The ethanologenic bacterium Zymomonas mobilis ZM4 is of special interest because it has a high ethanol yield. This is made possible by the two alcohol dehydrogenases (ADHs) present in Z. mobilis ZM4 (zmADHs), which shift the equilibrium of the reaction toward the synthesis of ethanol. They are metal-dependent enzymes: zinc for zmADH1 and iron for zmADH2. However, zmADH2 is inactivated by oxygen, thus implicating zmADH2 as the component of the cytosolic respiratory system in Z. mobilis. Here, we show crystal structures of zmADH2 in the form of an apo-enzyme and an NAD⁺-cofactor complex. The overall folding of the monomeric structure is very similar to those of other functionally related ADHs with structural variations around the probable substrate and NAD⁺ cofactor binding region. A dimeric structure is formed by the limited interactions between the two subunits with the bound NAD⁺ at the cleft formed along the domain interface. The catalytic iron ion binds near to the nicotinamide ring of NAD⁺, which is likely to restrict and locate the ethanol to the active site together with the oxidized Cys residue and several nonpolar bulky residues. The structures of the zmADH2 from the proficient ethanologenic bacterium Z. mobilis, with and without NAD⁺ cofactor, and modeling ethanol in the active site imply that there is a typical metal-dependent catalytic mechanism.     

Determination of the transglycosidation activity of NAD+ glycohydrolases with 4-(2'-alkyl-sulfanyl-vinyl)-pyridine derivatives generating chromophoric NAD+ analogs

The base exchange of nicotinamide with pyridine derivatives 1a-5a, catalyzed by pig brain NAD(+) glycohydrolase and ADP-ribosyl cyclase from Aplysia californica, generated the corresponding NAD(+) analogs 1b-5b. These analogs exhibited a high absorbance band in the visible region. The transglycosidation rate was determined by monitoring the absorbance increase. Among the tested derivatives, (E)-4-[2-(methylsulfanyl)-vinyl]-pyridine 1a was the most suitable substrate for pig brain NAD(+) glycohydrolase while 4-[1,3]-dithiolan-2-ylidenemethyl-pyridine 3a was the most efficient for ADP-ribosyl cyclase from A. californica.     

Dunnione protects against experimental cisplatin-induced nephrotoxicity by modulating NQO1 and NAD+ levels

Despite being an efficacious anticancer agent, the clinical utility of cisplatin is hindered by its cardinal side effects. This investigation aimed to appraise potential protective impact of dunnione, a natural naphthoquinone pigment with established NQO1 stimulatory effects, on cisplatin nephrotoxicity of rats. Dunnione was administered orally at 10 and 20?mg/kg doses for 4 d and a single injection of cisplatin was delivered at the second day. Renal histopathology, inflammatory/oxidative stress/apoptotic markers, kidney function, and urinary markers of renal injury were assessed. Dunnione repressed cisplatin-induced inflammation in the kidneys as indicated by decreased TNF-α/IL-1β levels, and reduced nuclear phosphorylated NF-κB p65. This agent also obviated cisplatin-invoked oxidative stress as elucidated by decreased MDA/GSH levels and increased SOD/CAT activities. Dunnione, furthermore, improved renal histological deteriorations as well as caspase-3 activities and terminal deoxynucleotidyl transferase (TUNEL) positive cells, the indicators of apoptosis. Moreover, it up-regulated nuclear Nrf2 and cytosolic haeme-oxygenase-1 (HO-1) and NQO1 levels; meanwhile, promoted NAD⁺/NADH ratios followed by enhancing the activities of Sirt1 and PARP1; and further attenuated nuclear acetylated NF-κB p65. Dunnione additionally declined cisplatin-evoked retrogression in renal function and upraise in urinary markers of glomerular and tubular injury as demonstrated by decreased serum urea and creatinine with simultaneous reductions in urinary excretions of collagen type IV, podocin, cystatin C, and retinol-binding protein (RBP). Altogether, these findings offer dunnione as a potential protective agent against cisplatin-induced nephrotoxicity in rats.     

Conserved water-mediated recognition and dynamics of NAD+ (carboxamide group) to hIMPDH enzyme: water mimic approach toward the design of isoform-selective inhibitor

Inosine monophosphate dehydrogenase (IMPDH) enzyme involves in GMP biosynthesis pathway. Type I hIMPDH is expressed at lower levels in all cells, whereas type II is especially observed in acute myelogenous leukemia, chronic myelogenous leukemia cancer cells, and 10?ns simulation of the IMP–NAD⁺ complex structures (PDB ID. 1B3O and 1JCN) have revealed the presence of a few conserved hydrophilic centers near carboxamide group of NAD⁺. Three conserved water molecules (W1, W, and W1′) in di-nucleotide binding pocket of enzyme have played a significant role in the recognition of carboxamide group (of NAD⁺) to D274 and H93 residues. Based on H-bonding interaction of conserved hydrophilic (water molecular) centers within IMP–NAD⁺-enzyme complexes and their recognition to NAD⁺, some covalent modification at carboxamide group of di-nucleotide (NAD⁺) has been made by substituting the –CONH₂group by –CONHNH₂ (carboxyl hydrazide group) using water mimic inhibitor design protocol. The modeled structure of modified ligand may, though, be useful for the development of antileukemic agent or it could be act as better inhibitor for hIMPDH-II.     

Decreased cADPR and increased NAD+ in the Cd38-/- mouse

CD38 is a type II glycoprotein that catalyzes the formation of cyclic ADP-ribose (cADPR), an intracellular calcium signalling molecule, from nicotinamide adenine dinucleotide (NAD⁺). Using a modified version of the fluorimetric cycling assay for cADPR which reduces between-subject variability, we report significant decreases in brain and lung cADPR, which although similar to previously published values, showed much less individual variation. The reduced variation within each group suggests that the range of cADPR is narrower than previously thought, and that the regulatory mechanisms controlling these levels are more finely tuned. We also report significant increases in brain, lung, and kidney NAD⁺ in the Cd38^−/− mouse, and provide the first experimental demonstration of the proximate relationship between CD38 and NAD⁺.     

Detection of an NAD+-dependent malic enzyme locus in the atlantic salmonSalmo salar and other salmonid fish

Electrophoretic studies of malate oxidoreductases routinely assess variation in two enzymes, malate dehydrogenase (EC 1.1.1.37) and malic enzyme (NADP⁺) (EC 1.1.1.40). By modification of the standard isozyme staining conditions for these enzymes, we have resolved a new NAD⁺-preferring, MgCl₂-requiring malic enzyme which is indicated to be EC 1.1.1.39. The enzyme was detected in 10 salmonid fish species of the generaSalmo, Salvelinus, andOnchoryhncus. Phenotypic variation indicates that the novel enzyme is tetrameric and coded by a single locus. Inheritance inS. salar follows a single-locus model and the phenotypes are unlinked to polymorphisms for_s MDH-3,4* and_m MEP-2*, two malate oxidoreductase loci previously shown to be variable in this species.This work was supported by a contract to E. V. from Fisheries and Oceans Canada, St. John's, Newfoundland, and a postgraduate award to W. C. J. from the Department of Education for Northern Ireland.     

Isolation and characterization of an NAD+-degrading bacterium PTX1 and its role in chromium biogeochemical cycle

Microorganisms can reduce toxic chromate to less toxic trivalent chromium [Cr(III)]. Besides Cr(OH)₃ precipitates, some soluble organo-Cr(III) complexes are readily formed upon microbial, enzymatic, and chemical reduction of chromate. However, the biotransformation of the organo-Cr(III) complexes has not been characterized. We have previously reported the formation of a nicotinamide adenine dinucleotide (NAD⁺)-Cr(III) complex after enzymatic reduction of chromate. Although the NAD⁺-Cr(III) complex was stable under sterile conditions, microbial cells were identified as precipitates in a non-sterile NAD⁺-Cr(III) solution after extended incubation. The most dominant bacterium PTX1 was isolated and assigned to Leifsonia genus by phylogenetic analysis of 16S rRNA gene sequence. PTX1 grew slowly on NAD⁺ with a doubling time of 17 h, and even more slowly on the NAD⁺-Cr(III) complex with an estimated doubling time of 35 days. The slow growth suggests that PTX1 passively grew on trace NAD⁺ dissociated from the NAD⁺-Cr(III) complex, facilitating further dissociation of the complex and formation of Cr(III) precipitates. Thus, organo-Cr(III) complexes might be an intrinsic link of the chromium biogeochemical cycle; they can be produced during chromate reduction and then further mineralized by microorganisms.     

Stereospecificity of hydride transfer in NAD+-catalyzed 2-deoxy-scyllo-inosose synthase, the key enzyme in the biosynthesis of 2-deoxystreptamine-containing aminocyclitol antibiotics

The key enzyme in the biosynthesis of clinically important aminocyclitol antibiotics is 2-deoxy-scyllo-inosose synthase (DOIS), which converts ubiquitous d-glucose 6-phosphate (G-6-P) into the specific carbocycle, 2-deoxy-scyllo-inosose with an aid of NAD(+)-NADH recycling. The NAD(+)-dependent first step of the DOIS reaction was examined in detail by the use of 6-phosphonate and 6-homophosphonate analogs of G-6-P. Both analogs showed competitive inhibition against the DOIS reaction with K(i) values of 1.3 and 2.8 mM, respectively, due to their inability for the subsequent phosphate elimination. Based on the direct spectrophotometric observation of NADH formed by the hydride transfer from 6-phosphonate to NAD(+), the stereospecificity of the hydride transfer in the DOIS reaction was analyzed with 6-[4-(2)H]phosphonate and was found to be pro-R specific.     

NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes

Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that is involved in DNA repair and activated by DNA damage. When activated, PARP-1 consumes NAD(+) to form ADP-ribose polymers on acceptor proteins. Extensive activation of PARP-1 leads to glycolytic blockade, energy failure, and cell death. These events have been postulated to result from NAD(+) depletion. Here, we used primary astrocyte cultures to directly test this proposal, utilizing the endogenous expression of connexin-43 hemichannels by astrocytes to manipulate intracellular NAD(+) concentrations. Activation of PARP-1 with the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) produced NAD(+) depletion, glycolytic blockade, and cell death. Cultures incubated in high (10mM) extracellular concentrations of NAD(+) after MNNG exposure showed normalization of intracellular NAD(+) concentrations. Repletion of intracellular NAD(+) in this manner completely restored glycolytic capacity and prevented cell death. These results suggest that NAD(+) depletion is the cause of glycolytic failure after PARP-1 activation.     

Extracellular NAD induces a rise in [Ca]i in activated human monocytes via engagement of P2Y1 and P2Y11 receptors

Extracellular nicotinamide adenine dinucleotide (NAD⁺) is known to increase the intracellular calcium concentration [Ca²⁺]_i in different cell types and by various mechanisms. Here we show that NAD⁺ triggers a transient rise in [Ca²⁺]_i in human monocytes activated with lipopolysaccharide (LPS), which is caused by a release of Ca²⁺ from IP₃-responsive intracellular stores and an influx of extracellular Ca²⁺. By the use of P2 receptor-selective agonists and antagonists we demonstrate that P2 receptors play a role in the NAD⁺-induced calcium response in activated monocytes. Of the two subclasses of P2 receptors (P2X and P2Y) the P2Y receptors were considered the most likely candidates, since they share calcium signaling properties with NAD⁺. The identification of P2Y₁ and P2Y₁₁ as receptor subtypes responsible for the NAD⁺-triggered increase in [Ca²⁺]_i was supported by several lines of evidence. First, specific P2Y₁ and P2Y₁₁ receptor antagonists inhibited the NAD⁺-induced increase in [Ca²⁺]_i. Second, NAD⁺ was shown to potently induce calcium signals in cells transfected with either subtype, whereas untransfected cells were unresponsive. Third, NAD⁺ caused an increase in [cAMP]_i, prevented by the P2Y₁₁ receptor-specific antagonist NF157.     

The histone deacetylases Rpd3 and Hst1 antagonistically regulate de novo NAD+ metabolism in the budding yeast Saccharomyces cerevisiae

NAD⁺ is a cellular redox cofactor involved in many essential processes. The regulation of NAD⁺ metabolism and the signaling networks reciprocally interacting with NAD⁺-producing metabolic pathways are not yet fully understood. The NAD⁺-dependent histone deacetylase (HDAC) Hst1 has been shown to inhibit de novo NAD⁺ synthesis by repressing biosynthesis of nicotinic acid (BNA) gene expression. Here, we alternatively identify HDAC Rpd3 as a positive regulator of de novo NAD⁺ metabolism in the budding yeast Saccharomyces cerevisiae. We reveal that deletion of RPD3 causes marked decreases in the production of de novo pathway metabolites, in direct contrast to deletion of HST1. We determined the BNA expression profiles of rpd3Δ and hst1Δ cells to be similarly opposed, suggesting the two HDACs may regulate the BNA genes in an antagonistic fashion. Our chromatin immunoprecipitation analysis revealed that Rpd3 and Hst1 mutually influence each other’s binding distribution at the BNA2 promoter. We demonstrate Hst1 to be the main deacetylase active at the BNA2 promoter, with hst1Δ cells displaying increased acetylation of the N-terminal tail lysine residues of histone H4, H4K5, and H4K12. Conversely, we show that deletion of RPD3 reduces the acetylation of these residues in an Hst1-dependent manner. This suggests that Rpd3 may function to oppose spreading of Hst1-dependent heterochromatin and represents a unique form of antagonism between HDACs in regulating gene expression. Moreover, we found that Rpd3 and Hst1 also coregulate additional targets involved in other branches of NAD⁺ metabolism. These findings help elucidate the complex interconnections involved in effecting the regulation of NAD⁺ metabolism.     

Early Zn2+-induced effects on membrane potential account for primary heavy metal susceptibility in tolerant and sensitive Arabidopsis species

Background and Aims

Uptake of heavy metals by plant root cells depends on electro-physiological parameters of the plasma membrane. In this study, responses of the plasma membrane in root cells were analysed where early reactions to the metal ion-induced stress are localized. Three different Arabidopsis species with diverse strategies of their adaptation to heavy metals were compared: sensitive Arabidopsis thaliana and tolerant A. halleri and A. arenosa.

Methods

Plants of A. thaliana Col-0 ecotype and plants of A. arenosa and A. halleri originating from natural metallicolous populations were exposed to high concentrations of Zn²⁺. Plants were tested for root growth rate, cellular tolerance, plant morphology and cell death in the root apex. In addition, the membrane potential (E_M) of mature cortical root cells and changes in the pH of the liquid culture media were measured.

Key Results

Primary roots of A. halleri and A. arenosa plants grew significantly better at increased Zn²⁺ concentrations than A. thaliana plants. Elevated Zn²⁺ concentrations in the culture medium induced rapid changes in E_M. The reaction was species-specific and concentration-dependent. Arabidopsis halleri revealed the highest insensitivity of the plasma membrane and the highest survival rate under prolonged treatment with extra-high concentrations. Plants were able to effectively adjust the pH in the control, but much less at Zn²⁺-induced lower pH.

Conclusions

The results indicate a similar mode of early reaction to Zn²⁺, but with different extent in tolerant and sensitive species of Arabidopsis. The sensitivity of A. thaliana and a high tolerance of A. halleri and A. arenosa were demonstrated. Plasma membrane depolarization was lowest in the hyperaccumulator A. halleri and highest in A. thaliana. This indicates that rapid membrane voltage changes are an excellent tool to monitor the effects of heavy metals.     

Influx of extracellular Ca2+ involved in jasmonic-acid-induced elevation of [Ca2+]cyt and JR1 expression in Arabidopsis thaliana

The changes in cytosolic Ca²⁺ levels play important roles in the signal transduction pathways of many environmental and developmental stimuli in plants and animals. We demonstrated that the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) of Arabidopsis thaliana leaf cells was induced by exogenous application of jasmonic acid (JA). The elevation of [Ca²⁺]_cyt was detected within 1 min after JA treatment by the fluorescence intensity using laser scanning confocal microscopy, and the elevated level of fluorescence was maintained during measuring time. With pretreatment of nifedipine (Nif), a nonpermeable L-type channel blocker, the fluorescence of [Ca²⁺]_cyt induced by JA was inhibited in a dose-dependent manner. In contrast, verapamil, another L-type channel blocker, had no significant effect. Furthermore, Nif repressed JA-induced gene expression of JR1 but verapamil did not. JA-induced gene expression could be mimicked by higher concentration of extracellular Ca²⁺. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an antagonist of calmodulin (CaM), blocked the JA induction of JR1 expression while W-5 [N-(6-aminohexyl)-1-naphthalenesulfonamide], its inactive antagonist, had no apparent effect. These data provide the evidence that the influx of extracellular Ca²⁺ through Nif sensitive plasma membrane Ca²⁺ channel may be responsible for JA-induced elevation of [Ca²⁺]_cyt and downstream gene expression, CaM may be also involved in JA signaling pathway.     

Purification and characterization of an NAD+-linked formaldehyde dehydrogenase from the facultative RuMP cycle methylotrophArthrobacter P1

WhenArthrobacter P₁ is grown on choline, betaine, dimethylglycine or sarcosine, an NAD⁺-dependent formaldehyde dehydrogenase is induced. This formaldehyde dehydrogenase has been purified using ammonium sulphate fractionation, anion exchange- and hydrophobic interaction chromatography. The molecular mass of the native enzyme was 115 kDa±10 kDa. Gel electrophoresis in the presence of sodium dodecyl sulphate indicated that the molecular mass of the subunit was 56 kDa±3 kDa, which is consistent with a dimeric enzyme structure. After ammonium sulphate fractionation the partially purified enzyme required the addition of a reducing reagent in the assay mixture for maximum activity. The enzyme was highly specific for its substrates and the K_m values were 0.10 and 0.80 mM for formaldehyde and NAD⁺, respectively. The enzyme was heat-stable at 50° C for at least 10 min and showed a broad pH optimum of 8.1 to 8.5. The addition of some metal-binding compounds and thiol reagents inhibited the enzyme activity.Abbreviation RuMP Ribulose monophosphate     

Intracellular ca2+ stores could participate to abscisic acid-induced depolarization and stomatal closure in Arabidopsis thaliana

In Arabidopsis thaliana cell suspension,abscisic acid (aBa) induces changes in cytosolic calcium concentration ([Ca²⁺]_cyt) which are the trigger for aBa-induced plasma membrane anion current activation, H⁺-aTPase inhibition, and subsequent plasma membrane depolarization. In the present study, we took advantage of this model to analyze the implication of intracellular Ca²⁺ stores in aBa signal transduction through electrophysiological current measurements, cytosolic Ca²⁺ activity measurements with the apoaequorin Ca²⁺ reporter protein and external pH measurement. Intracellular Ca²⁺ stores involvement was determined by using specific inhibitors of CICR channels: the cADP-ribose/ryanodine receptor (Br-cADPR and dantrolene) and of the inositol trisphosphate receptor ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122). In addition experiments were performed on epidermal strips of A. thaliana leaves to monitor stomatal closure in response to ABA in presence of the same pharmacology. Our data provide evidence that ryanodine receptor and inositol trisphosphate receptor could be involved in ABA-induced (1) Ca²⁺ release in the cytosol, (2) anion channel activation and H⁺-ATPase inhibition leading to plasma membrane depolarization and (3) stomatal closure. Intracellular Ca²⁺ release could thus contribute to the control of early events in the ABA signal transduction pathway in A. thaliana.     

Functional expression and molecular characterization of AtUBC2-1, a novel ubiquitin-conjugating enzyme (E2) from Arabidopsis thaliana

The first member of a novel subfamily of ubiquitin-conjugating E2-proteins was cloned from a cDNA library of Arabidopsis thaliana. Genomic blots indicate that this gene family (AtUBC2) consists of two members and is distinct from AtUBC1, the only other E2 enzyme known from this species to date (M.L. Sullivan and R.D. Vierstra, Proc. Natl. Acad. Sci. USA 86 (1989) 9861-9865). The cDNA sequence of AtUBC2-1 extends over 794 bp which would encode a protein of 161 amino acids and a calculated molecular mass of 18.25 kDa. The protein encoded by AtUBC2-1 is shown to accept ¹²⁵I-ubiquitin from wheat E1 enzymes, when expressed from Escherichia coli hosts as fusion protein carrying N-terminal extensions. It is deubiquitinated in the presence of lysine and, by these criteria, is considered a functional E2 enzyme.     

The overexpression in Arabidopsis thaliana of a Trichoderma harzianum gene that modulates glucosidase activity, and enhances tolerance to salt and osmotic stresses

Using the TrichoEST database, generated in a previous functional genomics project from the beneficial filamentous fungus Trichoderma harzianum, a gene named Thkel1, which codes for a putative kelch-repeat protein, was isolated and characterized. Silencing of this gene in T. harzianum leads to a reduction of glucosidase activity and mycelial growth under abiotic stress conditions. Expression of this gene in Arabidopsis enhances plant tolerance to salt and osmotic stresses, accompanied by an increase in glucosidase activity and a reduction of abscisic acid levels compared to those observed in wild-type plants. Data presented throughout this article suggest the high value of T. harzianum as a source of genes able to facilitate the achievement of producing plants resistant to abiotic stresses without alteration of their phenotype.