Overexpression of Human Mutant PANK2 Proteins Affects Development and Motor Behavior of Zebrafish Embryos.

Pantothenate Kinase-Associated Neurodegeneration (PKAN) is a genetic and early-onset neurodegenerative disorder characterized by iron accumulation in the basal ganglia. It is due to mutations in Pantothenate Kinase 2 (PANK2), an enzyme that catalyzes the phosphorylation of vitamin B5, first and essential step in coenzyme A (CoA) biosynthesis. Most likely, an unbalance of the neuronal levels of this important cofactor represents the initial trigger of the neurodegenerative process, yet a complete understanding of the connection between PANK2 malfunctioning and neuronal death is lacking. Most PKAN patients carry mutations in both alleles and a loss of function mechanism is proposed to explain the pathology. When PANK2 mutants were analyzed for stability, dimerization capacity, and enzymatic activity in vitro, many of them showed properties like the wild-type form. To further explore this aspect, we overexpressed the wild-type protein, two mutant forms with reduced kinase activity and two retaining the catalytic activity in zebrafish embryos and analyzed the morpho-functional consequences. While the wild-type protein had no effects, all mutant proteins generated phenotypes that partially resembled those observed in pank2 and coasy morphants and were rescued by CoA and vitamin B5 supplementation. The overexpression of PANK2 mutant forms appears to be associated with perturbation in CoA availability, irrespective of their catalytic activity.

Dehydropropylpantothenamide isolated by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of animal cells.

A new amide, named dehydropropylpantothenamide (1), was obtained by a co-culture of Nocardia tenerifensis IFM 10554T in the presence of the mouse macrophage-like cell line J774.1 in modified Czapek-Dox (mCD) medium. Compound 1 was synthesized from D-pantothenic acid calcium salt in 6 steps. The absolute configuration of natural compound 1 was determined by comparisons of the optical rotation and CD spectra of synthetic 1. In the present study, a new method for producing secondary metabolites was demonstrated using a "co-culture" in which the genus Nocardia was cultured in the presence of an animal cell line.

Pantothenic acid biosynthesis in the parasite Toxoplasma gondii: a target for chemotherapy.

Toxoplasma gondii is a major food pathogen and neglected parasitic infection that causes eye disease, birth defects, and fetal abortion and plays a role as an opportunistic infection in AIDS. In this study, we investigated pantothenic acid (vitamin B5) biosynthesis in T. gondii. Genes encoding the full repertoire of enzymes for pantothenate synthesis and subsequent metabolism to coenzyme A were identified and are expressed in T. gondii. A panel of inhibitors developed to target Mycobacterium tuberculosis pantothenate synthetase were tested and found to exhibit a range of values for inhibition of T. gondii growth. Two inhibitors exhibited lower effective concentrations than the currently used toxoplasmosis drug pyrimethamine. The inhibition was specific for the pantothenate pathway, as the effect of the pantothenate synthetase inhibitors was abrogated by supplementation with pantothenate. Hence, T. gondii encodes and expresses the enzymes for pantothenate synthesis, and this pathway is essential for parasite growth. These promising findings increase our understanding of growth and metabolism in this important parasite and highlight pantothenate synthetase as a new drug target.

Organic cofactors in the metabolism of Dehalococcoides mccartyi strains.

Dehalococcoides mccartyi strains are strictly anaerobic organisms specialized to grow with halogenated compounds as electron acceptor via a respiratory process. Their genomes are among the smallest known for free-living organisms, and the embedded gene set reflects their strong specialization. Here, we briefly review main characteristics of published Dehalococcoides genomes and show how genome information together with cultivation and biochemical experiments have contributed to our understanding of Dehalococcoides physiology and biochemistry. We extend this approach by the detailed analysis of cofactor metabolism in Dehalococcoides strain CBDB1. Dehalococcoides genomes were screened for encoded proteins annotated to contain or interact with organic cofactors, and the expression of these proteins was analysed by shotgun proteomics to shed light on cofactor requirements. In parallel, cultivation experiments testing for vitamin requirements showed that cyanocobalamin (vitamin B12), thiamine and biotin were essential supplements and that cyanocobalamin could be substituted by dicyanocobinamide and dimethylbenzimidazole. Dehalococcoides genome analysis, detection of single enzymes by shotgun proteomics and inhibition studies confirmed the expression of the biosynthetic pathways for pyridoxal-5-phosphate, flavin nucleotides, folate, S-adenosylmethionine, pantothenate and nicotinic acids in strain CBDB1. Haem/cytochromes, quinones and lipoic acids were not necessary for cultivation or dechlorination activity and no biosynthetic pathways were identified in the genomes.

Towards engineering increased pantothenate (vitamin B(5)) levels in plants.

Pantothenate (vitamin B(5)) is the precursor of the 4'-phosphopantetheine moiety of coenzyme A and acyl-carrier protein. It is made by plants and microorganisms de novo, but is a dietary requirement for animals. The pantothenate biosynthetic pathway is well-established in bacteria, comprising four enzymic reactions catalysed by ketopantoate hydroxymethyltransferase (KPHMT), L: -aspartate-alpha-decarboxylase (ADC), pantothenate synthetase (PS) and ketopantoate reductase (KPR) encoded by panB, panD, panC and panE genes, respectively. In higher plants, the genes encoding the first (KPHMT) and last (PS) enzymes have been identified and characterised in several plant species. Commercially, pantothenate is chemically synthesised and used in vitamin supplements, feed additives and cosmetics. Biotransformation is an attractive alternative production system that would circumvent the expensive procedures of separating racemic intermediates. We explored the possibility of manipulating pantothenate biosynthesis in plants. Transgenic oilseed rape (Brassica napus) lines were generated in which the E. coli KPHMT and PS genes were expressed under a strong constitutive CaMV35SS promoter. No significant change of pantothenate levels in PS transgenic lines was observed. In contrast plants expressing KPHMT had elevated pantothenate levels in leaves, flowers siliques and seed in the range of 1.5-2.5 fold increase compared to the wild type plant. Seeds contained the highest vitamin content, indicating that they might be the ideal target for production purposes.

Organisation of the pantothenate (vitamin B5) biosynthesis pathway in higher plants.

Pantothenate (vitamin B5) is the precursor for the biosynthesis of the phosphopantetheine moiety of coenzyme A and acyl carrier protein, and is synthesised in Escherichia coli by four enzymic reactions. Ketopantoate hydroxymethyltransferase (KPHMT) and pantothenate synthetase (PtS) catalyse the first and last steps, respectively. Two genes encoding KPHMT and one for PtS were identified in the Arabidopsis thaliana genome, and cDNAs for all three genes were amplified by PCR. The cDNAs were able to complement their respective E. coli auxotrophs, demonstrating that they encoded functional enzymes. Subcellular localisation of the proteins was investigated using green fluorescent protein (GFP) fusions and confocal microscopy. The two KPHMT-GFP fusion proteins were targeted exclusively to mitochondria, whereas PtS-GFP was found in the cytosol. This implies that there must be transporters for pathway intermediates. KPHMT enzyme activity could be measured in purified mitochondria from both pea leaves and Arabidopsis suspension cultures. We investigated whether Arabidopsis encoded homologues of the remaining two pantothenate biosynthesis enzymes from E. coli, l-aspartate-alpha-decarboxylase (ADC) and ketopantoate reductase (KPR). No homologue of ADC could be identified using either conventional blast or searches with the program fugue in which the structure of the E. coli ADC was compared to all the annotated proteins in Arabidopsis. ADC also appears to be absent from the genome of the yeast, Saccharomyces cerevisiae, by the same criteria. In contrast, a putative Arabidopsis oxidoreductase with some similarity to KPR was identified with fugue.

Pantothenate production in Escherichia coli K12 by enhanced expression of the panE gene encoding ketopantoate reductase.

Using gene replacement and transposon Tn5 mutagenesis, an Escherichia coli ilvC panE double mutant completely lacking ketopantoate reductase activity was isolated. This E. coli double mutant was employed to isolate the E. coli panE gene by genetic complementation. The E. coli panE gene is characterized by a 912 bp coding region, which specifies a protein of 303 amino acids with a deduced molecular mass of 33.8 kD. A panE expression plasmid carrying the panE gene under the control of the tac promotor was constructed. Introduction of the panE expression plasmid into E. coli resulted in a threefold increase in ketopantoate reductase activity. It was also shown that the enhanced panE expression in E. coli K12 led to 3.5-fold increase in pantothenate excretion. Pantothenate excretion could even be more enhanced when the growth medium was supplemented with ketopantoate.

Role of acetohydroxy acid isomeroreductase in biosynthesis of pantothenic acid in Salmonella typhimurium.

Structural genes have been identified for all of the enzymes involved in the biosynthesis of pantothenic acid in Salmonella typhimurium and Escherichia coli K-12, with the exception of ketopantoic acid reductase, which catalyzes the conversion of alpha-ketopantoate to pantoate. The acetohydroxy acid isomeroreductase from S. typhimurium efficiently bound alpha-ketopantoate (K(m) = 0.25 mM) and catalyzed its reduction at 1/20 the rate at which alpha-acetolactate was reduced. Since two enzymes could apparently participate in the synthesis of pantoate, a S. typhimurium ilvC8 strain was mutagenized to derive strains completely blocked in the conversion of alpha-ketopantoate to pantoate. Several isolates were obtained that grew in isoleucine-valine medium supplemented with either pantoate or pantothenate, but not in the same medium supplemented with alpha-ketopantoate or beta-alanine. The mutations that conferred pantoate auxotrophy (designated panE) to these isolates appeared to be clustered, but were not linked to panB or panC. All panE strains tested had greatly reduced levels of ketopantoic acid reductase (3 to 12% of the activity present in DU201). The capacity of the isomeroreductase to synthesize pantoate in vivo was assessed by determining the growth requirements of ilvC(+) derivatives of panE ilvC8 strains. These strains required either alpha-ketopantoate, pantoate, or pantothenate when the isomeroreductase was present at low levels; when the synthesis of isomeroreductase was induced, panE ilvC(+) strains grew in unsupplemented medium. These phenotypes indicate that a high level of isomeroreductase is sufficient for the synthesis of pantoate. panE ilvC(+) strains also grew in medium supplemented with lysine and methionine. This phenotype resembles that of some S. typhimurium ilvG mutants (e.g., DU501) which are partially blocked in the biosynthesis of coenzyme A and are limited for succinyl coenzyme A. panE ilvC(+) strains which lack the acetohydroxy acid synthases required only methionine for growth (in the presence of leucine, isoleucine, and valine). This and other evidence suggested that the synthesis of pantoic acid by isomeroreductase was blocked by the alpha-acetohydroxy acids and that pantoic acid synthesis was enhanced in the absence of these intermediates, even when the isomeroreductase was at low levels. panE ilvC(+) strains reverted to pantothenate independence. Several of these revertants were shown to have elevated isomeroreductase levels under noninduced and induced conditions; the suppressing mutation in each revertant was shown to be closely linked to ilvC by P22 transduction. This procedure presents a means for obtaining mutants with altered regulation of isomeroreductase.

Genetic and biochemical analyses of pantothenate biosynthesis in Escherichia coli and Salmonella typhimurium.

Pantothenate (pan) auxotrophs of Escherichia coli K-12 and Salmonella typhimurium LT2 were characterized by enzymatic and genetic analyses. The panB mutants of both organisms and the pan-6 ("panA") mutant of S. typhimurium are deficient in ketopantoate hydroxymethyltransferase, whereas the panC mutants lack pantothenate synthetase. panD mutants of E. coli K-12 were previously shown to be deficient in aspartate 1-decarboxylase. All mutants showed only a single enzyme defect. The finding that the pan-6 mutant was deficient in ketopantoate hydroxymethyltransferase indicates that the genetic lesion is a panB allele. The pan-6 mutant therefore is deficient in the utilization of alpha-ketoisovalerate rather than the synthesis of alpha-ketoisovalerate, as originally proposed. The order of the pan genes of E. coli K-12 was determined by phage P1-mediated three-factor crosses. The clockwise order was found to be aceF panB panD panC tonA on the genetic map of E. coli K-12. The three-factor crosses were greatly facilitated by use of a closely linked Tn10 transposon as the outside marker. We also found that supplementation of E. coli K-12 auxotrophs with a high concentration of pantothenate or beta-alanine increased the intracellular coenzyme A level two- to threefold above the normal level. Supplementation with pantoate or ketopantoate resulted in smaller increases.