Cloning, functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase

In this paper we report the cloning and full sequencing of S-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.50) cDNA from Vitis vinifera L. (VV) leaves, an enzyme belonging to the polyamine biosynthetic pathway, which appears to play an important role in the regulation of plant growth and development. The presence of two overlapping ORFs (tiny ORF and small ORF) upstream of the main ORF is reported in the Vitis cDNA. When the Vitis SAMDC cDNA was expressed in yeast without the two upstream ORFs, the resulting activity was about 50 times higher than the activity obtained with the full cDNA. These results demonstrated the strong regulatory activity of the tiny and small ORFs. RT-PCR expression analysis showed evidence of a similar mRNA level in all the tissues tested, with the exception of the petioles. The VV SAMDC was also modelled using its homologues from Solanum tuberosum and Homo sapiens as template. The present work confirmed, for the first time in a woody plant of worldwide economic interest such as grapevine, the presence of a regulatory mechanism of SAMDC, enzyme that has a well-established importance in the modulation of plant growth and development.     

Polyamines and Ca2+ Mediate Hyperosmolal Opening of the Blood-Brain Barrier: In Vitro Studies in Isolated Rat Cerebral Capillaries

We recently presented evidence that the reversible opening of the blood-brain barrier (BBB) by the infusion of 1.6 M mannitol into the rat internal carotid artery is mediated by a rapid stimulation of ornithine decarboxylase (ODC) activity and putrescine synthesis in cerebral capillaries. We have now investigated this hypothesis further, using isolated rat cerebral capillaries as an in vitro model of the BBB. The ODC activity of cerebral capillary preparations was enriched up to 15-fold over that of the cerebral homogenate. Hyperosmolal mannitol in physiological buffer evoked a rapid (less than 15 s), concentration- and time-dependent increase in capillary ODC activity and an accumulation of putrescine and spermidine which was blocked by the specific ODC inhibitor, alpha-difluoromethylornithine (DFMO, 10 mM). Mannitol (1 M), as well as 2 M urea, evoked a two- to fivefold increase in the temperature-sensitive influx of 45Ca2+ and uptake of horseradish peroxidase (HRP) and 2-deoxy-D-[1-3H]glucose (DG), but not alpha-[1-14C]aminoisobutyrate, during a 2-min incubation. DFMO (10 mM) abolished 1 M mannitol-mediated stimulation of 45Ca2+ influx and uptake of HRP and DG, whereas 1 mM putrescine replenished capillary polyamines and reversed the DFMO effects. Mannitol (1 M)-induced stimulation of ODC activity and membrane transport processes was Ca2+-dependent and verapamil- and nisoldipine-sensitive. Phorbol myristate acetate (PMA, 10 nM), a protein kinase C activator, also evoked a two- to threefold stimulation of 45Ca2+ transport and HRP and DG uptake. This PMA effect was abolished by DFMO, suggesting involvement of rapid, ODC-controlled polyamine synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison between the homocyclic aromatic metabolic pathways from plant-derived compounds by bacteria and fungi

Aromatic compounds derived from lignin are of great interest for renewable biotechnical applications. They can serve in many industries e.g. as biochemical building blocks for bioplastics or biofuels, or as antioxidants, flavor agents or food preservatives. In nature, lignin is degraded by microorganisms, which results in the release of homocyclic aromatic compounds. Homocyclic aromatic compounds can also be linked to polysaccharides, tannins and even found freely in plant biomass. As these compounds are often toxic to microbes already at low concentrations, they need to be degraded or converted to less toxic forms. Prior to ring cleavage, the plant- and lignin-derived aromatic compounds are converted to seven central ring-fission intermediates, i.e. catechol, protocatechuic acid, hydroxyquinol, hydroquinone, gentisic acid, gallic acid and pyrogallol through complex aromatic metabolic pathways and used as energy source in the tricarboxylic acid cycle. Over the decades, bacterial aromatic metabolism has been described in great detail. However, the studies on fungal aromatic pathways are scattered over different pathways and species, complicating a comprehensive view of fungal aromatic metabolism. In this review, we depicted the similarities and differences of the reported aromatic metabolic pathways in fungi and bacteria. Although both microorganisms share the main conversion routes, many alternative pathways are observed in fungi. Understanding the microbial aromatic metabolic pathways could lead to metabolic engineering for strain improvement and promote valorization of lignin and related aromatic compounds.     

Crystal structure of uncleaved L-aspartate-alpha-decarboxylase from Mycobacterium tuberculosis

L-aspartate-alpha-decarboxylase (ADC) is a critical regulatory enzyme in the pantothenate biosynthetic pathway and belongs to a small class of self-cleaving and pyruvoyl-dependent amino acid decarboxylases. The expression level of ADC in Mycobacterium tuberculosis (Mtb) was confirmed by cDNA analysis, immunoblotting with an anti-ADC polyclonal antibody using whole cell lysate and immunoelectron microscopy. The recombinant ADC proenzyme from Mycobacterium tuberculosis (MtbADC) was overexpressed in E. coli and the protein structure was determined at 2.99 A resolution. The proteins fold into the double-psi beta-barrel structure. The subunits of the two tetramers (there are eight ADC molecules in the asymmetric unit) form pseudo fourfold rotational symmetry, similar to the E. coli ADC proenzyme structure. As pantothenate is synthesized in microorganisms, plants, and fungi but not in animals, structure elucidation of Mtb ADC is of substantial interest for structure-based drug development.     

Molecular characterization of ura1, a mutant allele for orotidine-5'-monophosphate decarboxylase in Schizophyllum commune

Abstract The basis of the auxotrophic ural phenotype in Schizophyllum commune has been investigated. Two point mutations causing changes in conserved amino acid positions 62 (from lysine to glutamate) and 79 (from leucine to phenylalanine) most likely are the cause for the observed phenotype, whereas the overall gene structure was unchanged. Since reversion rates in this locus are extremely low, a single point mutation could not be expected to be the cause for the mutation. Besides the two point mutations expected to be induced by UV mutagenesis, the two alleles investigated from independently isolated strains differ by approximately 7% in nucleic acid sequence and about 3% in amino acid sequence, indicating a distant relationship between the strains used.     

Inhibition of enzymes of polyamine biosynthesis by substrate-like O-substituted hydroxylamines

The biogenic amines spermine, spermidine, and putrescine are essential factors of cell growth and differentiation. To inhibit pyridoxal-5"-phosphate dependent ornithine decarboxylase and pyruvate dependent S-adenosylmethionine decarboxylase, key enzymes of polyamine biosynthesis, a system of substrate-like O-substituted hydroxylamines is suggested. The best of these compounds were active at nanomolar concentrations. High potency and specificity of this type of inhibitors are discussed in terms of structural similarity of E–I and E–S complexes.     

Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells

Phosphatidylserine (PS) and phosphatidylethanolamine (PE) are metabolically related membrane aminophospholipids. In mammalian cells, PS is required for targeting and function of several intracellular signaling proteins. Moreover, PS is asymmetrically distributed in the plasma membrane. Although PS is highly enriched in the cytoplasmic leaflet of plasma membranes, PS exposure on the cell surface initiates blood clotting and removal of apoptotic cells. PS is synthesized in mammalian cells by two distinct PS synthases that exchange serine for choline or ethanolamine in phosphatidylcholine (PC) or PE, respectively. Targeted disruption of each PS synthase individually in mice demonstrated that neither enzyme is required for viability whereas elimination of both synthases was embryonic lethal. Thus, mammalian cells require a threshold amount of PS. PE is synthesized in mammalian cells by four different pathways, the quantitatively most important of which are the CDP-ethanolamine pathway that produces PE in the ER, and PS decarboxylation that occurs in mitochondria. PS is made in ER membranes and is imported into mitochondria for decarboxylation to PE via a domain of the ER [mitochondria-associated membranes (MAM)] that transiently associates with mitochondria. Elimination of PS decarboxylase in mice caused mitochondrial defects and embryonic lethality. Global elimination of the CDP-ethanolamine pathway was also incompatible with mouse survival. Thus, PE made by each of these pathways has independent and necessary functions. In mammals PE is a substrate for methylation to PC in the liver, a substrate for anandamide synthesis, and supplies ethanolamine for glycosylphosphatidylinositol anchors of cell-surface signaling proteins. Thus, PS and PE participate in many previously unanticipated facets of mammalian cell biology. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Induction of acetoacetate decarboxylase in Clostridium acetobutylicum

Abstract Factors that may initiate the biosynthesis of acetoacetate decarboxylase were investigated in resting cells of Clostridium acetobutylicum . Linear acids from C₁ to C₄ were inducers, whereas branched acids and linear acids from C₅ to C₇ were not inducers of acetoacetate decarboxylase biosynthesis. Induction of acetoacetate decarboxylase was maximal at pH 4.8 in the presence of acid concentrations comparable with those found during fermentation. In growth conditions repression of acetoacetate decarboxylase biosynthesis was found. This fact explains that acetone production by Clostridium acetobutylicum occurs when growth slows down.     

GABA-shunt enzymes activity in GH3 cells with reduced level of PMCA2 or PMCA3 isoform

GABA (γ-aminobutyric acid) is important neurotransmitter and regulator of endocrine functions. Its metabolism involves three enzymes: glutamate decarboxylase (GAD65 and GAD67), GABA aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). As many cellular processes GABA turnover can depend on calcium homeostasis, which is maintained by plasma membrane calcium ATPases (PMCAs). In excitable cells PMCA2 and PMCA3 isoforms are particularly important. In this study we focused on GABA-metabolizing enzymes expression and activity in rat anterior pituitary GH3 cells with suppressed expression of PMCA2 or PMCA3. We observed that PMCA3-reduced cells have increased GAD65 expression. Suppression of PMCA2 caused a decrease in total GAD and GABA-T activity. These results indicate that PMCA2 and PMCA3 presence may be an important regulatory factor in GABA metabolism. Results suggest that PMCA2 and PMCA3 function is rather related to regulation of GABA synthesis and degradation than supplying cells with metabolites, which can be potentially energetic source.     

Aggregation of human lymphoblastoid cells by tumor-promoting phorbol esters and dihydroteleocidin B

Human lymphoblastoid cells transformed by Epstein-Barr virus aggregated rapidly in the presence of tumor-promoting phorbol esters and dihydroteleocidin B. Cell aggregation was almost complete after incubation for 6 hours. In amounts of a few ng, they induced significant aggregation. Their abilities to aggregate cells could be measured quantitatively and correlated well with their effects in promoting skin tumors.