Regulation of colominic acid biosynthesis by temperature: role of cytidine 5'-monophosphate N-acetylneuraminic acid synthetase

Synthesis of colominic acid in Escherichia coli K-235 is strictly regulated by temperature. Evidence for the role of cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) synthetase in this regulation was obtained by measuring its level in E. coli grown at 20 and 37°C. No activity was found in E. coli grown at 20°C. CMP-Neu5Ac started to be quickly synthesized when bacteria grown at 20°C were transferred to 37°C and was halted when cells grown at 37°C were transferred to 20°C. These findings suggest that temperature regulates the synthesis of this enzyme and therefore the concentration of CMP-Neu5Ac necessary for the biosynthesis of colominic acid.     

Controlled acid hydrolysis of colominic acid under microwave irradiation

The hydrolysis of colominic acid under microwave irradiation was studied and compared with traditional heating methods. The microwave irradiation has several advantages over the heating method in the hydrolysis of colominic acid: (a) products with higher degrees of polymerization are obtained, (b) less lactone byproducts are observed, and (c) the hydrolytic rate is much faster. These advantages are probably due to the microwave effect. Oligosialic acids as the products of the acid hydrolysis of polysialic acid with conventional heating methods were fully lactonized, especially under the conditions of higher temperature and stronger acid.     

A heteromeric plastidic pyruvate kinase complex involved in seed oil biosynthesis in Arabidopsis

Glycolysis is a ubiquitous pathway thought to be essential for the production of oil in developing seeds of Arabidopsis thaliana and oil crops. Compartmentation of primary metabolism in developing embryos poses a significant challenge for testing this hypothesis and for the engineering of seed biomass production. It also raises the question whether there is a preferred route of carbon from imported photosynthate to seed oil in the embryo. Plastidic pyruvate kinase catalyzes a highly regulated, ATP-producing reaction of glycolysis. The Arabidopsis genome encodes 14 putative isoforms of pyruvate kinases. Three genes encode subunits alpha, beta(1), and beta(2) of plastidic pyruvate kinase. The plastid enzyme prevalent in developing seeds likely has a subunit composition of 4alpha4beta(1), is most active at pH 8.0, and is inhibited by Glu. Disruption of the gene encoding the beta(1) subunit causes a reduction in plastidic pyruvate kinase activity and 60% reduction in seed oil content. The seed oil phenotype is fully restored by expression of the beta(1) subunit-encoding cDNA and partially by the beta(2) subunit-encoding cDNA. Therefore, the identified pyruvate kinase catalyzes a crucial step in the conversion of photosynthate into oil, suggesting a preferred plastid route from its substrate phosphoenolpyruvate to fatty acids.     

Pipecolic acid biosynthesis in Rhizoctonia leguminicola. II. Saccharopine oxidase: a unique flavin enzyme involved in pipecolic acid biosynthesis

The fungal parasite Rhizoctonia leguminicola produces two indolizidine alkaloids, slaframine and swainsonine, of physiological interest. These alkaloids are biosynthesized from pipecolic acid which in turn is derived from L-lysine in this fungus as shown in the accompanying paper (Wickwire, B.M., Harris, C.M., Harris, T.M., and Broquist, H.P. (1989) J. Biol. Chem. 265, 14742-14747): L-lysine----saccharopine----delta 1----piperideine-6- carboxylate----pipecolate. This paper concerns the discovery, purification, and properties of a flavoenzyme, termed saccharopine oxidase, which carries out the oxidative cleavage of saccharopine as follows: Saccharopine + O2----delta 1-piperidine-6-carboxylate + glutamate + H2O2 The enzyme was purified 2,000-fold to homogeneity (polyacrylamide gel electrophoresis) in 14% yield from R. leguminicola mycelia, and had a native molecular mass of about 45,000 daltons by gel filtration (fast protein liquid chromatography Superose). Evidence for the presence of a flavin in the enzyme was drawn from these considerations: (a) the enzyme, while oxidatively cleaving saccharopine, concomitantly reduces 2,6-dichlorophenolindophenol; (b) the purified enzyme has a fluorescence spectrum typical of flavins; and (c) the enzyme requires oxygen and produces hydrogen peroxide. Good correlation was shown with purified saccharopine oxidase between disappearance of saccharopine with the concomitant appearance of delta 1-piperideine-6-carboxylate plus glutamate. The enzyme has a pH optimum about 6 and a Km for saccharopine of 0.128 mM. The enzyme apparently exists in R. leguminicola to shunt saccharopine, a major lysine metabolite, into a secondary pathway of lysine metabolism leading to pipecolate and subsequently to slaframine and swainsonine.     

The genetic fine structure of the complex locus aro3 involved in early aromatic amino acid biosynthesis in Schizosaccharomyces pombe.

Summary The complex locus aro3 of Schizosaccharomyces pombe was subjected to genetical fine structure analysis. By comparing the complementation map and the meiotic recombination map, the aro3 locus could be subdivided into the five adjacent subregions A, B, C, D and E. Out of 115 aro3 alleles, 26 nonsense alleles and 30 missense alleles could be identified by the criteria of nonsense suppressor sensitivity and leakiness, respectively. Most alleles with a pleiotropic complementation pattern are of the nonsense type. We conclude from the polarity of the complementation patterns characterising the nonsense alleles that the translation direction proceeds from subregion A to subregion E. Antipolar effects in complementation are more frequent than in the analogous system of the arom gene cluster of Neurospora crassa.This work formed part of a Ph.D. thesis submitted to the University of Bern     

Inhibition of biosynthesis of Saccharomyces cerevisiae sugar transport system by tunicamycin.

Tunicamycin apparently inhibited the biosynthesis of glucose, galactose, and maltose transport systems in Saccharomyces cerevisiae. Under the conditions used, the antibiotic also blocked the biosynthesis of invertase, a well-known yeast glycoprotein, as well as the glycosylation of a marker mannoprotein of the yeast cell wall. However, the antibiotic did not affect certain proteins which did not contain carbohydrate. It seems, therefore, that these sugar carriers are glycoproteins.     

A tryptophan C-methyltransferase involved in streptonigrin biosynthesis in Streptomyces flocculus.

A C-methyltransferase that catalyses the transfer of a methyl group from S-adenosylmethionine to C-3 of tryptophan, resulting in beta-methyltryptophan, has been identified in cell-free extracts of streptonigrin-producing Streptomyces flocculus. The absolute configuration of the product was shown to be (2S,3R)-beta-methyltryptophan by high-pressure liquid chromatography and reactivity with D- and L-amino acid oxidases. In shake culture, maximum specific activity occurs after S. flocculus enters stationary phase, but before significant streptonigrin accumulates.     

Fractionation studies of the enzyme complex involved in teichoic acid synthesis.

The membrane-bound enzymes participating in the syntheses of the teichoic acid main chain and linkage unit have been solubilized with Triton X-100 and fractionated by sucrose density gradient centrifugation. Two main fractions were obtained: a heavy fraction, containing enzymes effecting synthesis of the main chain attached to the linkage unit, which was associated with only a small amount of lipid, and a light fraction which was rich in prenyl phosphate and catalyzed only linkage-unit synthesis. The separation by density was not based entirely on polypeptide chain length, as some of the shortest chains appeared in the denser fractions and some relatively high-molecular-weight peptides occurred in the lightest fraction. High activity for linkage-unit synthesis was observed in a fraction containing only a few peptides. Addition of ficaprenyl phosphate to the enzyme preparations had no stimulatory effect. It is concluded that the enzymes for main-chain and linkage unit syntheses frm one or more fairly tightly associated complexes and that polyprenyl phosphate is an integral firmly bound component of the complex in which the linkage unit is synthesized.     

Inhibitory effects of tunicamycin on procollagen biosynthesis and secretion

Chick embryo cells were briefly exposed to the antibiotic, tunicamycin. Pre-exposed cells, compared to control cultures, showed a severe, progressive inhibition of the incorporation of glucosamine and mannose into total cellular macromolecules. Inhibition of the incorporation of glycine, leucine and proline was also progressive but not as marked as for the carbohydrates. Cellular secretion of all macromolecules was severely impaired. while comparison of the procollagens showed no difference in their subunit size or in their degree of glycosylation; the intracellular content of procollagen polypeptides was similar for both types of cells. In vitro studies showed that tunicamycin selectively inhibited glucosamine, but not mannose, incorporation into macromolecules. The composite results indicate that tunicamycin effectively inhibits protein synthesis, protein glycosylation and protein secretion in chick embryo cells.     

Effect of retinoic acid on chondrocyte glycosaminoglycan biosynthesis.

The effect of retinoic acid on glycosaminoglycan biosynthesis was investigated in rat costal cartilage chondrocytes in vitro. At levels of 10^?9 to 10^?8m retinoic acid, ³⁵SO₄ uptake into glycosaminoglycans was reduced 50%. At these low levels of retinoic acid there was no evidence of lysosomal enzyme release. The results are explained best in terms of modification of glycosaminoglycan synthesis, rather than accelerated degradation. Retinoic acid selectively modified the incorporation of ³⁵SO₄ or [¹⁴C]glucosamine into individual glycosaminoglycans fractions under the conditions studied. The relative incorporation of radiolabeled precursor into heparan sulfate (and/or) heparin increased three- to fourfold. The relative incorporation of radiolabeled precursor remained constant for chondroitin 6-sulfate, whereas incorporation into chondroitin 4-sulfate and chondroitin (and/or) hyaluronic acid decreased. Under the conditions studied, retinoic acid did not appear to be cytotoxic and did exhibit selective control over glycosaminoglycan biosynthesis. It is suggested that the decreased incorporation of ³⁵SO₄ into glycosaminoglycans at hypervitaminosis A levels of retinol may be accounted for by the presence of low levels of retinoic acid, a naturally occurring metabolite.     

Cloning and functional characterization of Phaeodactylum tricornutum front-end desaturases involved in eicosapentaenoic acid biosynthesis.

Phaeodactylum tricornutum is an unicellular silica-less diatom in which eicosapentaenoic acid accumulates up to 30% of the total fatty acids. This marine diatom was used for cloning genes encoding fatty acid desaturases involved in eicosapentaenoic acid biosynthesis. Using a combination of PCR, mass sequencing and library screening, the coding sequences of two desaturases were identified. Both protein sequences contained a cytochrome b5 domain fused to the N-terminus and the three histidine clusters common to all front-end fatty acid desaturases. The full length clones were expressed in Saccharomyces cerevisiae and characterized as Delta5- and Delta6-fatty acid desaturases. The substrate specificity of each enzyme was determined and confirmed their involvement in eicosapentaenoic acid biosynthesis. Using both desaturases in combination with the Delta6-specific elongase from Physcomitrella patens, the biosynthetic pathways of arachidonic and eicosapentaenoic acid were reconstituted in yeast. These reconstitutions indicated that these two desaturases functioned in the omega3- and omega6-pathways, in good agreement with both routes coexisting in Phaeodactylum tricornutum. Interestingly, when the substrate selectivity of each enzyme was determined, both desaturases converted the omega3- and omega6-fatty acids with similar efficiencies, indicating that none of them was specific for either the omega3- or the omega6-pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.     

Effect of tunicamycin and cycloheximide on the secretion of acid hydrolases from I-cell cultured fibroblasts.

I-cell cultures fibroblasts secrete excessive amounts of N-acetyl-beta-D-hexosaminidase and alpha-L-fucosidase into the culture media as compared with normal fibroblasts. Addition of tunicamycin or cyd [14C]leucine (40--50%) into trichloroacetic acid-precipitable material decreased the secretion of these I-cell hydrolases to normal values within 24 h, but had no effect on the secretion of acid hydrolases from normal fibroblasts. These results indicate that I-cell cultured fibroblasts secrete at least two types of acid hydrolases: one is tunicamycin- and cycloheximide-sensitive and constitutes the greater proportion of the secreted hydrolases, and a smaller proportion is insensitive to tunicamycin and cycloheximide, similar t9 the acid hydrolases secreted by normal cultured fibroblasts.