Glutamate, Ornithine, Arginine, Proline, and Polyamine Metabolic Interactions: The Pathway Is Regulated at the Post-Transcriptional Level

The metabolism of glutamate into ornithine, arginine, proline, and polyamines is a major network of nitrogen-metabolizing pathways in plants, which also produces intermediates like nitric oxide, and γ-aminobutyric acid (GABA) that play critical roles in plant development and stress. While the accumulations of intermediates and the products of this network depend primarily on nitrogen assimilation, the overall regulation of the interacting sub-pathways is not well understood. We tested the hypothesis that diversion of ornithine into polyamine biosynthesis (by transgenic approach) not only plays a role in regulating its own biosynthesis from glutamate but also affects arginine and proline biosynthesis. Using two high putrescine producing lines of Arabidopsis thaliana (containing a transgenic mouse ornithine decarboxylase gene), we studied the: (1) effects of exogenous supply of carbon and nitrogen on polyamines and pools of soluble amino acids; and, (2) expression of genes encoding key enzymes in the interactive pathways of arginine, proline and GABA biosynthesis as well as the catabolism of polyamines. Our findings suggest that: (1) the overall conversion of glutamate to arginine and polyamines is enhanced by increased utilization of ornithine for polyamine biosynthesis by the transgene product; (2) proline and arginine biosynthesis are regulated independently of polyamines and GABA biosynthesis; (3) the expression of most genes (28 that were studied) that encode enzymes of the interacting sub-pathways of arginine and GABA biosynthesis does not change even though overall biosynthesis of Orn from glutamate is increased several fold; and (4) increased polyamine biosynthesis results in increased assimilation of both nitrogen and carbon by the cells

Coenzyme Q10 deficiencies: pathways in yeast and humans.

Coenzyme Q (ubiquinone or CoQ) is an essential lipid that plays a role in mitochondrial respiratory electron transport and serves as an important antioxidant. In human and yeast cells, CoQ synthesis derives from aromatic ring precursors and the isoprene biosynthetic pathway. Saccharomyces cerevisiae coq mutants provide a powerful model for our understanding of CoQ biosynthesis. This review focusses on the biosynthesis of CoQ in yeast and the relevance of this model to CoQ biosynthesis in human cells. The COQ1-COQ11 yeast genes are required for efficient biosynthesis of yeast CoQ. Expression of human homologs of yeast COQ1-COQ10 genes restore CoQ biosynthesis in the corresponding yeast coq mutants, indicating profound functional conservation. Thus, yeast provides a simple yet effective model to investigate and define the function and possible pathology of human COQ (yeast or human gene involved in CoQ biosynthesis) gene polymorphisms and mutations. Biosynthesis of CoQ in yeast and human cells depends on high molecular mass multisubunit complexes consisting of several of the COQ gene products, as well as CoQ itself and CoQ intermediates. The CoQ synthome in yeast or Complex Q in human cells, is essential for de novo biosynthesis of CoQ. Although some human CoQ deficiencies respond to dietary supplementation with CoQ, in general the uptake and assimilation of this very hydrophobic lipid is inefficient. Simple natural products may serve as alternate ring precursors in CoQ biosynthesis in both yeast and human cells, and these compounds may act to enhance biosynthesis of CoQ or may bypass certain deficient steps in the CoQ biosynthetic pathway

Biosynthesis of α-Tocopherol and Plastoquinone-9 in spinach chloroplasts

Prenylation and methylation reaction in al biosynthesis is localized in the envelope membranes of the chloroplasts, while PQ-9 biosynthesis takes place in the envelope membranes and also in the thylakoid membranes. The sequence in a-T biosynthesis in spinach is (see also Figure 1): Homogentisate + Phytyl-PP —> Me-6-PQH?—> 2,3-Me2PQH?—>γ J ->a T ; for the PQ-9 biosynthesis it is: Homogentisate + Solanesyf-PP4-> Me-6-SQH2—> PQH2

Aspergillus westerdijkiae polyketide synthase gene “aoks1” is involved in the biosynthesis of ochratoxin A

OchratoxinA (OTA) is a potential nephrotoxic, teratogenic, immunogenic, hepatotoxic and carcinogenic mycotoxin, produced by Aspergillus westerdijkiae NRRL 3174. Herein we describe the characterization of a putative OTA-polyketide synthasegene “aoks1”, cloned by using gene walking approach. The predicted amino acid sequence of the 2 kb clone display 34–60% similarities to different polyketide synthasegenes including lovastatine biosynthesis gene “lovb” in A. terreus, compactin biosynthesis gene “mlcA” in Penicillium citrinum and OTA biosynthesis gene “otapksPN” in P. nordicum. Based on the reverse transcription PCR and kinetic secondary metabolites production studies, aoks1 expression was found to be associated with OTA biosynthesis. Further a mutant, in which the aoks1gene was inactivated by Escherichia coli hygromycin B phosphotransferase gene, lost the capacity to produce OTA, but still producing mellein. To our knowledge this report describes for the first time characterization of a gene involved in OTA biosynthesis, with the information about mellein which was proposed in the literature to be an intermediate OTA. This study also suggests that aoks1 may be the second polyketide synthase gene required for OTA biosynthesis in A. westerdijkiae NRRL 3174

Limits of aerobic metabolism in cancer cells

Cancer cells exhibit high rates of glycolysis and glutaminolysis. Glycolysis can provide energy and glutaminolysis can provide carbon for anaplerosis and reductive carboxylation to citrate. However, all these metabolic requirements could be in principle satisfied from glucose. Here we investigate why cancer cells do not satisfy their metabolic demands using aerobic biosynthesis from glucose. Based on the typical composition of a mammalian cell we quantify the energy demand and the OxPhos burden of cell biosynthesis from glucose. Our calculation demonstrates that aerobic growth from glucose is feasible up to a minimum doubling time that is proportional to the OxPhos burden and inversely proportional to the mitochondria OxPhos capacity. To grow faster cancer cells must activate aerobic glycolysis for energy generation and uncouple NADH generation from biosynthesis. To uncouple biosynthesis from NADH generation cancer cells can synthesize lipids from carbon sources that do not produce NADH in their catabolism, including acetate and the amino acids glutamate, glutamine, phenylalanine and tyrosine. Finally, we show that cancer cell lines have an OxPhos capacity that is insufficient to support aerobic biosynthesis from glucose. We conclude that selection for high rate of biosynthesis implies a selection for aerobic glycolysis and uncoupling biosynthesis from NADH generation

Maritime pine PpMYB8 directly co-regulates secondary cell wall architecture and the associated Phe-biosynthesis pathway

Plants rely on the biosynthesis of L-Phenylalanine as building block for the synthesis of proteins but also as precursor for a tremendous range of plant-derived compounds essential for its grown, development and defense. Polymerization of secondary cell wall in trees involves the massive biosynthesis, among others, of the Phe-derived compound lignin. Thus, these plants require an accurate metabolic coordination between Phe and lignin biosynthesis to ensure its normal development. We have here identified that the pine arogenate dehydratase, whose enzyme activity limits the biosynthesis of Phe in plants, is transcriptionally regulated through direct interaction with PpMyb8. We have also shown that this transcription factor is directly involve in secondary cell wall biogenesis and cell death processes. Together these results indicate that a single transcription factor coordinates lignin accumulation and the proper biosynthesis of its essential precursor L-Phe.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Gene-to-metabolite network for biosynthesis of lignans in MeJA-elicited Isatis indigotica hairy root cultures.

Root and leaf tissue of Isatis indigotica shows notable anti-viral efficacy, and are widely used as "Banlangen" and "Daqingye" in traditional Chinese medicine. The plants' pharmacological activity is attributed to phenylpropanoids, especially a group of lignan metabolites. However, the biosynthesis of lignans in I. indigotica remains opaque. This study describes the discovery and analysis of biosynthetic genes and AP2/ERF-type transcription factors involved in lignan biosynthesis in I. indigotica. MeJA treatment revealed differential expression of three genes involved in phenylpropanoid backbone biosynthesis (IiPAL, IiC4H, Ii4CL), five genes involved in lignan biosynthesis (IiCAD, IiC3H, IiCCR, IiDIR, and IiPLR), and 112 putative AP2/ERF transcription factors. In addition, four intermediates of lariciresinol biosynthesis were found to be induced. Based on these results, a canonical correlation analysis using Pearson's correlation coefficient was performed to construct gene-to-metabolite networks and identify putative key genes and rate-limiting reactions in lignan biosynthesis. Over-expression of IiC3H, identified as a key pathway gene, was used for metabolic engineering of I. indigotica hairy roots, and resulted in an increase in lariciresinol production. These findings illustrate the utility of canonical correlation analysis for the discovery and metabolic engineering of key metabolic genes in plants

RORγ is a targetable master regulator of cholesterol biosynthesis in a cancer subtype.

Tumor subtype-specific metabolic reprogrammers could serve as targets of therapeutic intervention. Here we show that triple-negative breast cancer (TNBC) exhibits a hyper-activated cholesterol-biosynthesis program that is strongly linked to nuclear receptor RORγ, compared to estrogen receptor-positive breast cancer. Genetic and pharmacological inhibition of RORγ reduces tumor cholesterol content and synthesis rate while preserving host cholesterol homeostasis. We demonstrate that RORγ functions as an essential activator of the entire cholesterol-biosynthesis program, dominating SREBP2 via its binding to cholesterol-biosynthesis genes and its facilitation of the recruitment of SREBP2. RORγ inhibition disrupts its association with SREBP2 and reduces chromatin acetylation at cholesterol-biosynthesis gene loci. RORγ antagonists cause tumor regression in patient-derived xenografts and immune-intact models. Their combination with cholesterol-lowering statins elicits superior anti-tumor synergy selectively in TNBC. Together, our study uncovers a master regulator of the cholesterol-biosynthesis program and an attractive target for TNBC

Further characterization of Dothistromin genes in the fungal forest pathogen Dothistroma septosporum : a thesis presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Genetics at Massey University, Palmerston North, New Zealand

Dothistroma septosporum is a forest pathogen that causes a disease called Dothistroma needle blight. The symptoms are thought to be due to the accumulation of dothistromin toxin produced by D. septosporum. Dothistromin is characterized as a difuranoanthraquinone and shows remarkable similarity to the aflatoxin (AF) and sterigmatocystin (ST) precursor versicolorin B. The similar structure to AF/ST suggests that dothistromin biosynthesis shares biosynthetic steps with the AF/ST pathway. The AF gene cluster in Aspergillus parasiticus and ST gene cluster in A. nidulans have been well characterized. Nine putative dothistromin biosynthetic genes have been identified. One of them, dotA was previously characterized by gene disruption and shown to have a similar function to homologous genes in AF/ST biosynthesis. Two additional putative dothistromin biosynthetic genes, pksA and epoA, were characterized by gene disruption in this study. The inability of the pksA mutants to produce dothistromin indicated that the pksA is a key gene in dothistromin biosynthesis. The feeding of intermediates confirmed that pksA gene product is required for a very early step of dothistromin biosynthesis. The pksA mutants also showed reduced sporulation compared to wildtype, suggesting a relationship between dothistromin production and sporulation. The epoA gene replacements were also obtained successfully by homologous recombination. Both Southern blot and northern hybridization confirmed that the epoA gene was disrupted. However, the epoA mutants did not show any difference to the wild type in three analyses (growth rate, sporulation rate, dothistromin biosynthesis). However it was not possible to rule out a role for EpoA at a very late stage of dothistromin biosynthesis. RACE analysis of the nine identified dothistromin genes characterized the transcription start and stop sites of the genes. Analyzing the putative regulatory protein binding motifs in the untranscribed region of the genes provided clues about the regulation of dothistromin biosynthesis and suggested there might be an aflR-like gene that governs dothistromin biosynthesis. Both the pksA gene disruption and the RACE results suggested that the dothistromin biosynthetic pathway is homologous to that of AF/ST biosynthesis. Further work on the dothistromin gene cluster will help us to understand the evolution of fungal toxin gene clusters

A Green Chemical Approach for the Synthesis of Gold Nanoparticles: Characterization and Mechanistic Aspect

This IRCSET-EMPOWER (Irish Research Council for Science, Engineering and Technology Postdoctoral Research Grant) project aims to improve current methodology for the synthesis of metal nanoparticles (NPs). The development of efficient methodology for metal nanomaterials synthesis is an economical and environmental challenge. While the current methods for NPs synthesis are often energy-intensive and involve toxic chemicals, NPs biosynthesis can be carried on at circumneutral pH and mild temperature, resulting in low cost and environmental impact. Nanomaterial biosynthesis has been already observed in magnetotactic bacteria, diatoms, and S-layer bacteria, however, controlled NPs biosynthesis is a relatively new area of research with considerable potential for development. A thorough understanding of the biochemical mechanism involved in NPs biosynthesis is needed, before biosynthetic methods can be economically competitive. The analysis and identification of active species in the nucleation and growth of metal NPs is a daunting task, due to the complexity of the microbial system. This project work focuses on the controlled biosynthesis of gold NPs by fungal microorganisms and aims to determine the biochemical mechanism involved in nucleation and growth of the particles