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.

Arabidopsis mutant analysis and gene regulation define a nonredundant role for glutamate dehydrogenase in nitrogen assimilation.

Glutamate dehydrogenase (GDH) is ubiquitous to all organisms, yet its role in higher plants remains enigmatic. To better understand the role of GDH in plant nitrogen metabolism, we have characterized an Arabidopsis mutant (gdh1-1) defective in one of two GDH gene products and have studied GDH1 gene expression. GDH1 mRNA accumulates to highest levels in dark-adapted or sucrose-starved plants, and light or sucrose treatment each repress GDH1 mRNA accumulation. These results suggest that the GDH1 gene product functions in the direction of glutamate catabolism under carbon-limiting conditions. Low levels of GDH1 mRNA present in leaves of light-grown plants can be induced by exogenously supplied ammonia. Under such conditions of carbon and ammonia excess, GDH1 may function in the direction of glutamate biosynthesis. The Arabidopsis gdh-deficient mutant allele gdh1-1 cosegregates with the GDH1 gene and behaves as a recessive mutation. The gdh1-1 mutant displays a conditional phenotype in that seedling growth is specifically retarded on media containing exogenously supplied inorganic nitrogen. These results suggest that GDH1 plays a nonredundant role in ammonia assimilation under conditions of inorganic nitrogen excess. This notion is further supported by the fact that the levels of mRNA for GDH1 and chloroplastic glutamine synthetase (GS2) are reciprocally regulated by light.

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.

Regulation of mitochondrial pyruvate dehydrogenase activity by tau protein kinase I/glycogen synthase kinase 3beta in brain.

According to the amyloid hypothesis for the pathogenesis of Alzheimer disease, beta-amyloid peptide (betaA) directly affects neurons, leading to neurodegeneration and tau phosphorylation. In rat hippocampal culture, betaA exposure activates tau protein kinase I/glycogen synthase kinase 3beta (TPKI/GSK-3beta), which phosphorylates tau protein into Alzheimer disease-like forms, resulting in neuronal death. To elucidate the mechanism of betaA-induced neuronal death, we searched for substrates of TPKI/GSK-3beta in a two-hybrid system and identified pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA in mitochondria. PDH was phosphorylated and inactivated by TPKI/GSK-3beta in vitro and also in betaA-treated hippocampal cultures, resulting in mitochondrial dysfunction, which would contribute to neuronal death. In cholinergic neurons, betaA impaired acetylcholine synthesis without affecting choline acetyltransferase activity, which suggests that PDH is inactivated by betaA-induced TPKI/GSK-3beta. Thus, TPKI/GSK-3beta regulates PDH and participates in energy metabolism and acetylcholine synthesis. These results suggest that TPKI/GSK-3beta plays a key role in the pathogenesis of Alzheimer disease.

Localization of 17beta-hydroxysteroid dehydrogenase and characterization of testosterone in the brain of the male frog.

Several enzymes involved in the formation of steroids of the pregnene and pregnane series have been identified in the brain, but the biosynthesis of testosterone has never been reported in the central nervous system. In the present study, we have investigated the distribution and bioactivity of 17beta-hydroxysteroid dehydrogenase (17beta-HSD) (EC 1.1.1.62; a key enzyme that is required for the formation of testosterone and estradiol) in the brain of the male frog Rana ridibunda. By using an antiserum against human type I placental 17beta-HSD, immunoreactivity was localized in a discrete group of ependymal glial cells bordering the telencephalic ventricles. HPLC analysis of telencephalon and hypothalamus extracts combined with testosterone radioimmunoassay revealed the existence of two peaks coeluting with testosterone and 5alpha-dihydrotestosterone. After HPLC purification, testosterone was identified by gas chromatography/mass spectrometry. Incubation of telencephalon slices with [3H]pregnenolone resulted in the formation of metabolites which coeluted with progesterone, 17alpha-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone. The newly synthesized steroid comigrating with testosterone was selectively immunodetected by using testosterone antibodies. These data indicate that 17beta-HSD is expressed in a subpopulation of gliocytes in the frog telencephalon and that telencephalic cells are capable of synthesizing various androgens, including dehydroepiandrosterone, androstenedione, testosterone, and 5alpha-dihydrotestosterone.

Retrotransposon insertion induces an isozyme of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

The insertion of the blood retrotransposon into the untranslated region of exon 7 of the sn-glycerol-3-phosphate dehydrogenase-encoding gene (Gpdh) in Drosophila melanogaster induces a GPDH isozyme-GPDH-4-and alters the pattern of expression of the three normal isozymes-GPDH-1 to GPDH-3. The process of transcript terminus formation inside the retrotransposon insertion reduces the level of the Gpdh transcript that contains exon 8 and increases the level of the transcript that contains exons 1-7. The induced GPDH-4 isozyme is a translation product of the three transcripts that contain fragments of the blood retrotransposon. The mechanism of mutagenesis by the blood insertion is postulated to involve the pause or termination of transcription within the blood sequence, which in turn is caused by the interference of a DNA-binding protein with the RNA polymerase. Thus, we show the formation of a new functional GPDH protein by the insertion of a transposable element and discuss the evolutionary significance of this phenomenon.

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

The isocitrate dehydrogenase of Escherichia coli, which lacks the Rossmann fold common to other dehydrogenases, displays a 7000-fold preference for NADP over NAD (calculated as the ratio of kcat/Km). Guided by x-ray crystal structures and molecular modeling, site-directed mutagenesis has been used to introduce six substitutions in the adenosine binding pocket that systematically shift coenzyme preference toward NAD. The engineered enzyme displays an 850-fold preference for NAD over NADP, which exceeds the 140-fold preference displayed by a homologous NAD-dependent enzyme. Of the six mutations introduced, only one is identical in all related NAD-dependent enzyme sequences--strict adherence to homology as a criterion for replacing these amino acids impairs function. Two additional mutations at remote sites improve performance further, resulting in a final mutant enzyme with kinetic characteristics and coenzyme preference comparable to naturally occurring homologous NAD-dependent enzymes.

The vertebrate alcohol dehydrogenase system: variable class II type form elucidates separate stages of enzymogenesis.

A mixed-class alcohol dehydrogenase has been characterized from avian liver. Its functional properties resemble the classical class I type enzyme in livers of humans and animals by exhibiting low Km and kcat values with alcohols (Km = 0.7 mM with ethanol) and low Ki values with 4-methylpyrazole (4 microM). These values are markedly different from corresponding parameters of class II and III enzymes. In contrast, the primary structure of this avian liver alcohol dehydrogenase reveals an overall relationship closer to class II and to some extent class III (69 and 65% residue identities, respectively) than to class I or the other classes of the human alcohol dehydrogenases (52-61%), the presence of an insertion (four positions in a segment close to position 120) as in class II but in no other class of the human enzymes, and the presence of several active site residues considered typical of the class II enzyme. Hence, the avian enzyme has mixed-class properties, being functionally similar to class I, yet structurally similar to class II, with which it also clusters in phylogenetic trees of characterized vertebrate alcohol dehydrogenases. Comparisons reveal that the class II enzyme is approximately 25% more variable than the "variable" class I enzyme, which itself is more variable than the "constant" class III enzyme. The overall extreme, and the unusual chromatographic behavior may explain why the class II enzyme has previously not been found outside mammals. The properties define a consistent pattern with apparently repeated generation of novel enzyme activities after separate gene duplications.

Molecular basis of human mitochondrial very-long-chain acyl-CoA dehydrogenase deficiency causing cardiomyopathy and sudden death in childhood.

beta-Oxidation of long-chain fatty acids provides the major source of energy in the heart. Defects in enzymes of the beta-oxidation pathway cause sudden, unexplained death in childhood, acute hepatic encephalopathy or liver failure, skeletal myopathy, and cardiomyopathy. Very-long-chain acyl-CoA dehydrogenase [VLCAD; very-long-chain-acyl-CoA:(acceptor) 2,3-oxidoreductase, EC 1.3.99.13] catalyzes the first step in beta-oxidation. We have isolated the human VLCAD cDNA and gene and determined the complete nucleotide sequences. Polymerase chain reaction amplification of VLCAD mRNA and genomic exons defined the molecular defects in two patients with VLCAD deficiency who presented with unexplained cardiac arrest and cardiomyopathy. In one, a homozygous mutation in the consensus dinucleotide of the donor splice site (g+1-->a) was associated with universal skipping of the prior exon (exon 11). The second patient was a compound heterozygote, with a missense mutation, C1837-->T, changing the arginine at residue 613 to tryptophan on one allele and a single base deletion at the intron-exon 6 boundary as the second mutation. This initial delineation of human mutations in VLCAD suggests that VLCAD deficiency reduces myocardial fatty acid beta-oxidation and energy production and is associated with cardiomyopathy and sudden death in childhood.

Sequence analysis of a mannitol dehydrogenase cDNA from plants reveals a function for the pathogenesis-related protein ELI3.

Mannitol is the most abundant sugar alcohol in nature, occurring in bacteria, fungi, lichens, and many species of vascular plants. Celery (Apium graveolens L.), a plant that forms mannitol photosynthetically, has high photosynthetic rates thought to results from intrinsic differences in the biosynthesis of hexitols vs. sugars. Celery also exhibits high salt tolerance due to the function of mannitol as an osmoprotectant. A mannitol catabolic enzyme that oxidizes mannitol to mannose (mannitol dehydrogenase, MTD) has been identified. In celery plants, MTD activity and tissue mannitol concentration are inversely related. MTD provides the initial step by which translocated mannitol is committed to central metabolism and, by regulating mannitol pool size, is important in regulating salt tolerance at the cellular level. We have now isolated, sequenced, and characterized a Mtd cDNA from celery. Analyses showed that Mtd RNA was more abundant in cells grown on mannitol and less abundant in salt-stressed cells. A protein database search revealed that the previously described ELI3 pathogenesis-related proteins from parsley and Arabidopsis are MTDs. Treatment of celery cells with salicylic acid resulted in increased MTD activity and RNA. Increased MTD activity results in an increased ability to utilize mannitol. Among other effects, this may provide an additional source of carbon and energy for response to pathogen attack. These responses of the primary enzyme controlling mannitol pool size reflect the importance of mannitol metabolism in plant responses to divergent types of environmental stress.

Complex genetic findings in a female patient with pyruvate dehydrogenase complex deficiency: Null mutations in the PDHX gene associated with unusual expression of the testis-specific PDHA2 gene in her somatic cells

Human pyruvate dehydrogenase complex (PDC) catalyzes a key step in the generation of cellular energy and is composed by three catalytic elements (E1, E2, E3), one structural subunit (E3-binding protein), and specific regulatory elements, phosphatases and kinases (PDKs, PDPs). The E1α subunit exists as two isoforms encoded by different genes: PDHA1 located on Xp22.1 and expressed in somatic tissues, and the intronless PDHA2 located on chromosome 4 and only detected in human spermatocytes and spermatids. We report on a young adult female patient who has PDC deficiency associated with a compound heterozygosity in PDHX encoding the E3-binding protein. Additionally, in the patient and in all members of her immediate family, a full-length testis-specific PDHA2 mRNA and a 5′UTR-truncated PDHA1 mRNA were detected in circulating lymphocytes and cultured fibroblasts, being bothmRNAs translated into full-length PDHA2 and PDHA1 proteins, resulting in the co-existence of both PDHA isoforms in somatic cells.Moreover, we observed that DNA hypomethylation of a CpG island in the coding region of PDHA2 gene is associatedwith the somatic activation of this gene transcription in these individuals. This study represents the first natural model of the de-repression of the testis-specific PDHA2 gene in human somatic cells, and raises some questions related to the somatic activation of this gene as a potential therapeutic approach for most forms of PDC deficiency.

Walking performance, oxygen uptake kinetics and resting muscle pyruvate dehydrogenase complex activity in peripheral arterial disease

In the present study, we tested the hypothesis that walking intolerance in intermittent claudication (IC) is related to both slowed whole body oxygen uptake (Vo(2)) kinetics and altered activity of the active fraction of the pyruvate dehydrogenase complex (PDCa) in skeletal muscle. Ten patients with IC and peripheral arterial disease [ankle/brachial index (ABI) = 0.73 +/- 0.13] and eight healthy controls (ABI = 1. 17 +/- 0.13) completed three maximal walking tests. From these tests, averaged estimates of walking time, peak Vo(2) and the time constant of Vo(2) (tau) during submaximal walking were obtained. A muscle sample was taken from the gastrocnemius medialis muscle at rest and analysed for PDCa and several other biochemical variables. Walking time and peak Vo(2) were approx. 50 % lower in patients with IC than controls, and tau was 2-fold higher (P < 0.05). r was significantly correlated with walking time (r = -0.72) and peak Vo(2) (r = -0.66) in patients with IC, but not in controls. PDCa was not significantly lower in patients with IC than controls; however, PDCa tended to be correlated with tau (r = -0.56, P = 0.09) in patients with IC, but not in controls (r = -0.14). A similar correlation was observed between resting ABI and tau (r = -0.63, P = 0.05) in patients with IC. These data suggest that the impaired Vo(2) kinetics contributes to walking intolerance in IC and that, within a group of patients with IC, differences in Vo(2) kinetics might be partly linked to differences in muscle carbohydrate oxidation.

Crystallization and preliminary X-ray analysis of sulfite dehydrogenase from Stargeya novella

Crystals of purified heterodimeric sulfite dehydrogenase from Starkeya novella have been grown using vapour diffusion. X-ray diffraction data have been collected from crystals of the native protein at lambda=1.0 Angstrom and close to the iron absorption edge at lambda=1.737 Angstrom. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a=97.5, b=92.5, c=55.9 Angstrom. Native data have been recorded to 1.8 Angstrom resolution and Fe-edge data to 2.5 Angstrom.

Insulin and Oleate Promote Translocation of Inosine-5' Monophosphate Dehydrogenase to Lipid Bodies

In the present study we identify inosine-5' monophosphate dehydrogenase (IMPDH), a key enzyme in de novo guanine nucleotide biosynthesis, as a novel lipid body-associated protein. To identify new targets of insulin we performed a comprehensive 2-DE analysis of P-32-labelled proteins isolated from 3T3-L1 adipocytes (Hill et al. J Biol Chem 2000; 275: 24313-24320). IMPDH was identified by liquid chromatography/tandem mass spectrometry as a protein which was phosphorylated in a phosphatidylinositol (PI) 3-kinase-dependent manner upon insulin treatment. Although insulin had no significant effect on IMPDH activity, we observed translocation of IMPDH to lipid bodies following insulin treatment. Induction of lipid body formation with oleic acid promoted dramatic redistribution of IMPDH to lipid bodies, which appeared to be in contact with the endoplasmic reticulum, the site of lipid body synthesis and recycling. Inhibition of PI 3-kinase blocked insulin- and oleate-induced translocation of IMPDH and reduced oleate-induced lipid accumulation. However, we found no evidence of oleate-induced IMPDH phosphorylation, suggesting phosphorylation and translocation may not be coupled events. These data support a role for IMPDH in the dynamic regulation of lipid bodies and fatty acid metabolism and regulation of its activity by subcellular redistribution in response to extracellular factors that modify lipid metabolism.