Two mitochondrial group I introns in a metazoan, the sea anemone Metridium senile: one intron contains genes for subunits 1 and 3 of NADH dehydrogenase.

Mitochondrial genes for cytochrome c oxidase subunit I (COI) and NADH dehydrogenase subunit 5 (ND5) of the sea anemone Metridium senile (phylum Cnidaria) each contain a group I intron. This is in contrast to the reported absence of introns in all other metazoan mtDNAs so far examined. The ND5 intron is unusual in that it ends with A and contains two genes (ND1 and ND3) encoding additional subunits of NADH dehydrogenase. Correctly excised ND5 introns are not circularized but are precisely cleaved near their 3' ends and polyadenylylated to provide bicistronic transcripts of ND1 and ND3. COI introns, which encode a putative homing endonuclease, circularize, but in a way that retains the entire genome-encoded intron sequence (other group I introns are circularized with loss of a short segment of the intron 5' end). Introns were detected in the COI and ND5 genes of other sea anemones, but not in the COI and ND5 genes of other cnidarians. This suggests that the sea anemone mitochondrial introns may have been acquired relatively recently.

d-β-Hydroxybutyrate protects neurons in models of Alzheimer's and Parkinson's disease

The heroin analogue 1-methyl-4-phenylpyridinium, MPP+, both in vitro and in vivo, produces death of dopaminergic substantia nigral cells by inhibiting the mitochondrial NADH dehydrogenase multienzyme complex, producing a syndrome indistinguishable from Parkinson's disease. Similarly, a fragment of amyloid protein, Aβ1–42, is lethal to hippocampal cells, producing recent memory deficits characteristic of Alzheimer's disease. Here we show that addition of 4 mM d-β-hydroxybutyrate protected cultured mesencephalic neurons from MPP+ toxicity and hippocampal neurons from Aβ1–42 toxicity. Our previous work in heart showed that ketone bodies, normal metabolites, can correct defects in mitochondrial energy generation. The ability of ketone bodies to protect neurons in culture suggests that defects in mitochondrial energy generation contribute to the pathophysiology of both brain diseases. These findings further suggest that ketone bodies may play a therapeutic role in these most common forms of human neurodegeneration.

Molecular evidence for an African origin of the Hawaiian endemic Hesperomannia (Asteraceae)

Identification of the progenitors of plants endemic to oceanic islands often is complicated by extreme morphological divergence between island and continental taxa. This is especially true for the Hawaiian Islands, which are 3,900 km from any continental source. We examine the origin of Hesperomannia, a genus of three species endemic to Hawaii that always have been placed in the tribe Mutisieae of the sunflower family. Phylogenetic analyses of representatives from all tribes in this family using the chloroplast gene ndhF (where ndhF is the ND5 protein of chloroplast NADH dehydrogenase) indicate that Hesperomannia belongs to the tribe Vernonieae. Phylogenetic comparisons within the Vernonieae using sequences of both ndhF and the internal transcribed spacer regions of nuclear ribosomal DNA reveal that Hesperomannia is sister to African species of Vernonia. Long-distance dispersal northeastward from Africa to southeast Asia and across the many Pacific Ocean island chains is the most likely explanation for this unusual biogeographic connection. The 17- to 26-million-year divergence time between African Vernonia and Hesperomannia estimated by the DNA sequences predates the age of the eight existing Hawaiian Islands. These estimates are consistent with an hypothesis that the progenitor of Hesperomannia arrived at one of the low islands of the Hawaiian-Emperor chain between the late Oligocene and mid-Miocene when these islands were above sea level. Subsequent to its arrival the southeast Pacific island chains served as steppingstones for dispersal to the existing Hawaiian Islands.

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mm) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mm) with respect to NAD+. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.

The NAD(P)H Dehydrogenase in Barley Thylakoids Is Photoactivatable and Uses NADPH as well as NADH1

An improved light-dependent assay was used to characterize the NAD(P)H dehydrogenase (NDH) in thylakoids of barley (Hordeum vulgare L.). The enzyme was sensitive to rotenone, confirming the involvement of a complex I-type enzyme. NADPH and NADH were equally good substrates for the dehydrogenase. Maximum rates of activity were 10 to 19 μmol electrons mg−1 chlorophyll h−1, corresponding to about 3% of linear electron-transport rates, or to about 40% of ferredoxin-dependent cyclic electron-transport rates. The NDH was activated by light treatment. After photoactivation, a subsequent light-independent period of about 1 h was required for maximum activation. The NDH could also be activated by incubation of the thylakoids in low-ionic-strength buffer. The kinetics, substrate specificity, and inhibitor profiles were essentially the same for both induction strategies. The possible involvement of ferredoxin:NADP+ oxidoreductase (FNR) in the NDH activity could be excluded based on the lack of preference for NADPH over NADH. Furthermore, thenoyltrifluoroacetone inhibited the diaphorase activity of FNR but not the NDH activity. These results also lead to the conclusion that direct reduction of plastoquinone by FNR is negligible.

Association of Ferredoxin-NADP Oxidoreductase with the Chloroplastic Pyridine Nucleotide Dehydrogenase Complex in Barley Leaves1

Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2′-monophosphoadenosine-5′-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2′-monophosphoadenosine-5′-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex.

Partial Purification and Characterization of the Maize Mitochondrial Pyruvate Dehydrogenase Complex1

The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1β-, E1α-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg2+. Michaelis constants for the substrates were 47, 3, 77, and 1 μm for pyruvate, coenzyme A (CoA), NAD+, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD+ and CoA, and the inhibition constants were 15 and 47 μm, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg2+.

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

The identification of a 9-cis retinol dehydrogenase in the mouse embryo reveals a pathway for synthesis of 9-cis retinoic acid

The ligand-controlled retinoic acid (RA) receptors and retinoid X receptors are important for several physiological processes, including normal embryonic development, but little is known about how their ligands, all-trans and 9-cis RA, are generated. Here we report the identification of a stereo-specific 9-cis retinol dehydrogenase, which is abundantly expressed in embryonic tissues known to be targets in the retinoid signaling pathway. The membrane-bound enzyme is a member of the short-chain alcohol dehydrogenase/reductase superfamily, able to oxidize 9-cis retinol into 9-cis retinaldehyde, an intermediate in 9-cis RA biosynthesis. Analysis by nonradioactive in situ hybridization in mouse embryos shows that expression of the enzyme is temporally and spatially well controlled during embryogenesis with prominent expression in parts of the developing central nervous system, sensory organs, somites and myotomes, and several tissues of endodermal origin. The identification of this enzyme reveals a pathway in RA biosynthesis, where 9-cis retinol is generated for subsequent oxidation to 9-cis RA.

Mitochondrial localization of a NADR-dependent isocitrate dehydrogenase isoenzyme by using the green fluorescent protein as a marker

In this work, we describe the isolation of a new cDNA encoding an NADP-dependent isocitrate dehydrogenase (ICDH). The nucleotide sequence in its 5′ region gives a deduced amino acid sequence indicative of a targeting peptide. However, even if this cDNA clearly encodes a noncytosolic ICDH, it is not possible to say from the targeting peptide sequence to which subcellular compartment the protein is addressed. To respond to this question, we have transformed tobacco plants with a construct containing the entire targeting signal-encoding sequence in front of a modified green fluorescent protein (GFP) gene. This construct was placed under the control of the cauliflower mosaic virus 35S promoter, and transgenic tobacco plants were regenerated. At the same time, and as a control, we also have transformed tobacco plants with the same construct but lacking the nucleotide sequence corresponding to the ICDH-targeting peptide, in which the GFP is retained in the cytoplasm. By optical and confocal microscopy of leaf epiderm and Western blot analyses, we show that the putative-targeting sequence encoded by the cDNA addresses the GFP exclusively into the mitochondria of plant cells. Therefore, we conclude that this cDNA encodes a mitochondrial ICDH.

In Azotobacter vinelandii, the E1 subunit of the pyruvate dehydrogenase complex binds fpr promoter region DNA and ferredoxin I

In Azotobacter vinelandii, deletion of the fdxA gene that encodes a well characterized seven-iron ferredoxin (FdI) is known to lead to overexpression of the FdI redox partner, NADPH:ferredoxin reductase (FPR). Previous studies have established that this is an oxidative stress response in which the fpr gene is transcriptionally activated to the same extent in response to either addition of the superoxide propagator paraquat to the cells or to fdxA deletion. In both cases, the activation occurs through a specific DNA sequence located upstream of the fpr gene. Here, we report the identification of the A. vinelandii protein that binds specifically to the paraquat activatable fpr promoter region as the E1 subunit of the pyruvate dehydrogenase complex (PDHE1), a central enzyme in aerobic respiration. Sequence analysis shows that PDHE1, which was not previously suspected to be a DNA-binding protein, has a helix–turn–helix motif. The data presented here further show that FdI binds specifically to the DNA-bound PDHE1.

CP12 provides a new mode of light regulation of Calvin cycle activity in higher plants

CP12 is a small nuclear encoded chloroplast protein of higher plants, which was recently shown to interact with NAD(P)H–glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13), one of the key enzymes of the reductive pentosephosphate cycle (Calvin cycle). Screening of a pea cDNA library in the yeast two-hybrid system for proteins that interact with CP12, led to the identification of a second member of the Calvin cycle, phosphoribulokinase (PRK; EC 2.7.1.19), as a further specific binding partner for CP12. The exchange of cysteines for serines in CP12 demonstrate that interaction with PRK occurs at the N-terminal peptide loop of CP12. Size exclusion chromatography and immunoprecipitation assays reveal the existence of a stable 600-kDa PRK/CP12/GAPDH complex in the stroma of higher plant chloroplasts. Its stoichiometry is proposed to be of two N-terminally dimerized CP12 molecules, each carrying one PRK dimer on its N terminus and one A2B2 complex of GAPDH subunits on the C-terminal peptide loop. Incubation of the complex with NADP or NADPH, in contrast to NAD or NADH, causes its dissociation. Assays with the stromal 600-kDa fractions in the presence of the four different nicotinamide-adenine dinucleotides indicate that PRK activity depends on complex dissociation and might be further regulated by the accessible ratio of NADP/NADPH. From these results, we conclude that light regulation of the Calvin cycle in higher plants is not only via reductive activation of different proteins by the well-established ferredoxin/thioredoxin system, but in addition, by reversible dissociation of the PRK/CP12/GAPDH complex, mediated by NADP(H).

Glyceraldehyde-3-phosphate dehydrogenase: Nuclear translocation participates in neuronal and nonneuronal cell death

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels increase in particulate fractions in association with cell death in HEK293 cells, S49 cells, primary thymocytes, PC12 cells, and primary cerebral cortical neuronal cultures. Subcellular fractionation and immunocytochemistry reveal that this increase primarily reflects nuclear translocation. Nuclear GAPDH is tightly bound, resisting extraction by DNase or salt treatment. Treating primary thymocytes, PC12 cells, and primary cortical neurons with antisense but not sense oligonucleotides to GAPDH prevents cell death. Because cell-death-associated nuclear translocation of GAPDH and antisense protection occur in multiple neuronal and nonneuronal systems, we propose that GAPDH is a general mediator of cell death and uses nuclear translocation as a signaling mechanism.

NMR characterization of altered lignins extracted from tobacco plants down-regulated for lignification enzymes cinnamylalcohol dehydrogenase and cinnamoyl-CoA reductase

Homologous antisense constructs were used to down-regulate tobacco cinnamyl-alcohol dehydrogenase (CAD; EC 1.1.1.195) and cinnamoyl-CoA reductase (CCR; EC 1.2.1.44) activities in the lignin monomer biosynthetic pathway. CCR converts activated cinnamic acids (hydroxycinnamoyl–SCoAs) to cinnamaldehydes; cinnamaldehydes are then reduced to cinnamyl alcohols by CAD. The transformations caused the incorporation of nontraditional components into the extractable tobacco lignins, as evidenced by NMR. Isolated lignin of antisense-CAD tobacco contained fewer coniferyl and sinapyl alcohol-derived units that were compensated for by elevated levels of benzaldehydes and cinnamaldehydes. Products from radical coupling of cinnamaldehydes, particularly sinapaldehyde, which were barely discernible in normal tobacco, were major components of the antisense-CAD tobacco lignin. Lignin content was reduced in antisense-CCR tobacco, which displayed a markedly reduced vigor. That lignin contained fewer coniferyl alcohol-derived units and significant levels of tyramine ferulate. Tyramine ferulate is a sink for the anticipated build-up of feruloyl–SCoA, and may be up-regulated in response to a deficit of coniferyl alcohol. Although it is not yet clear whether the modified lignins are true structural components of the cell wall, the findings provide further indications of the metabolic plasticity of plant lignification. An ability to produce lignin from alternative monomers would open new avenues for manipulation of lignin by genetic biotechnologies.

Conformation of coenzyme pyrroloquinoline quinone and role of Ca2+ in the catalytic mechanism of quinoprotein methanol dehydrogenase

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ)Ca2+, (PQQH)Ca2+, and (PQQH2)Ca2+ complexes as well as the x-ray structure of (PQQ)Ca2+ bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)Ca2+ complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of Ca2+, nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303–CO2−general-base removal of the methanol proton of the (PQQ)Ca2+O(H)CH3 complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the Ca2+ moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the pKa of methanol substrate,; and (iii) polarize the oxygen at C-5.