The presence of an active S-adenosylmethionine decarboxylase gene increases the growth defect observed in Saccharomyces cerevisiae mutants unable to synthesize putrescine, spermidine, and spermine.

Saccharomyces cerevisiae spe1 delta SPE2 mutants (lacking ornithine decarboxylase) and spe1 delta spe2 delta mutants (lacking both ornithine decarboxylase and S-adenosylmethionine decarboxylase) are equally unable to synthesize putrescine, spermidine, and spermine and require spermidine or spermine for growth in amine-free media. The cessation of growth, however, occurs more rapidly in spe1 delta SPE2 cells than in SPE1 spe2 delta or spe1 delta spe2 delta cells. Since spe1 delta SPE2 cells can synthesize decarboxylated adenosylmethionine (dcAdoMet), these data indicate that dcAdoMet may be toxic to amine-deficient cells.     

Arginine decarboxylase is a component activity of the multifunctional enzyme putrescine synthase in cucumber seedlings

A homogenous PreParation of Putrescine synthase, the versatile multifunctional enzyme involved in agmatine →Putrescine conversion inCucumis sativus was found to catalyze enzymatic decarboxylation of arginine also. Similarly, the Purified arginine decarboxylase mediated the comPonent as well as the comPlete set of couPled reactions harboured by Putrescine synthase. Both the enzyme PreParations exhibited identical electroPhoretic and chromatograPhic behaviour and were immunologically indistinguishable. All the enzymic activities are stabilized concurrently by feeding arginine to the intact seedlings. Therefore, it is concluded that the multifunctional Putrescine synthase inCucumis sativus seedlings also harbours arginine decarboxylase activity unlike its counterPart inLathyrus sativus.     

Construction of an Escherichia coli strain unable to synthesize putrescine, spermidine, or cadaverine: characterization of two genes controlling lysine decarboxylase.

We have previously described a polyamine-deficient strain of Escherichia coli that contained deletions in speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), and speD (adenosylmethionine decarboxylase). Although this strain completely lacked putrescine and spermidine, it was still able to grow at a slow rate indefinitely on amine-deficient media. However, these cells contained some cadaverine (1,5-diaminopentane). To rule out the possibility that the presence of cadaverine permitted the growth of this strain, we isolated a mutant (cadA) that is deficient in cadaverine biosynthesis, namely, a mutant lacking lysine decarboxylase, and transduced this cadA gene into the delta (speA-speB) delta speC delta D strain. The resultant strain had essentially no cadaverine but showed the same phenotypic characteristics as the parent. Thus, these results confirm our previous findings that the polyamines are not essential for the growth of E. coli or for the replication of bacteriophages T4 and T7. We have mapped the cadA gene at 92 min; the gene order is mel cadA groE ampA purA. A regulatory gene for lysine decarboxylase (cadR) was also obtained and mapped at 46 min; the gene order is his cdd cadR fpk gyrA.     

Findings of trigonelline demethylating enzyme activity in various organisms and some properties of the enzyme from hog liver

Trigonelline demethylating enzyme activity was found widely in animals, plants and microorganisms. Very high enzyme activity of this enzyme was detected in hog liver. Properties of the hog liver enzyme were investigated. Optimum pH for the enzymic reaction was observed at 8.5. The Km value for trigonelline was calculated at 2.77 mM. Addition of any cofactor is not required for the reaction The enzyme activity was inhibited by heavy metal ions. The reaction product was identified as nicotinic acid. Proposed enzyme reaction mechanism and the role of this enzyme in biosynthesis and metabolism of NAD are discussed.     

Control of ornithine decarboxylase in Chinese hamster ovary cells by polyamines. Translational inhibition of synthesis and acceleration of degradation of the enzyme by putrescine, spermidine, and spermine

We have recently isolated, without using any inhibitors, a mutant of Chinese hamster ovary cell line which greatly overproduces ornithine decarboxylase in serum-free culture. Addition of polyamines (putrescine, spermidine, or spermine, 10 microM) or ornithine (1 mM), the precursor of polyamines, to the culture medium of these cells caused a rapid and extensive decay of ornithine decarboxylase activity. At the same time the activity of S-adenosylmethionine decarboxylase showed a less pronounced decrease. Notably, the polyamine concentrations used were optimal for growth of the cells and caused no perturbation of general protein synthesis. Spermidine and spermine appeared to be the principal regulatory amines for both enzymes, but also putrescine, if accumulated at high levels in the cells, was capable of suppressing ornithine decarboxylase activity. The amount of ornithine decarboxylase protein (as measured by radioimmunoassay) declined somewhat more slowly than the enzyme activity, but no more than 10% of the loss of activity could be ascribed to post-translational modifications or inhibitor interaction. Some evidence for inactivation through ornithine decarboxylase-antizyme complex formation was obtained. Gel electrophoretic determinations of the [35S]methionine-labeled ornithine decarboxylase revealed a rapid reduction in the synthesis and acceleration in the degradation of the enzyme after polyamine additions. No decrease in the amounts of the two ornithine decarboxylase-mRNA species, hybridizable to a specific cDNA, was detected, suggesting that polyamines depressed ornithine decarboxylase synthesis by selectively inhibiting translation of the message.     

H-colchicine binding : Failure to detect any binding to soluble proteins from various lower organisms

No ³H-colchicine binding could be detected to post-ribosomal supernatants from Schizosaccharomyces pombe, Chlamydomonas reinhardii, Tetrahymena pyriformis, pea root tips, or Zea mays coleoptiles. However, under the same conditions, ³H-colchicine bound to supernatants from mouse brain and unfertilised sea urchin eggs. A protein with similar characteristics to sea urchin and mouse brain tubulins in terms of its molecular weight and elution properties from DEAE-cellulose columns has been detected in supernatants from the yeast S. pombe. Similar results (unpublished) have been obtained for both Zea mays and Tetrahymena. This protein is tentatively identified as tubulin. The lack of binding of ³H-colchicine by supernatants from lower organisms is consistent with the variable sensitivity of nuclear division to colchicine, and with the possible evolution of the colchicine-binding site of tubulin.     

Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate

Oxalyl-coenzyme A decarboxylase is a thiamin diphosphate-dependent enzyme that plays an important role in the catabolism of the highly toxic compound oxalate. We have determined the crystal structure of the enzyme from Oxalobacter formigenes from a hemihedrally twinned crystal to 1.73 A resolution and characterized the steady-state kinetic behavior of the decarboxylase. The monomer of the tetrameric enzyme consists of three alpha/beta-type domains, commonly seen in this class of enzymes, and the thiamin diphosphate-binding site is located at the expected subunit-subunit interface between two of the domains with the cofactor bound in the conserved V-conformation. Although oxalyl-CoA decarboxylase is structurally homologous to acetohydroxyacid synthase, a molecule of ADP is bound in a region that is cognate to the FAD-binding site observed in acetohydroxyacid synthase and presumably fulfils a similar role in stabilizing the protein structure. This difference between the two enzymes may have physiological importance since oxalyl-CoA decarboxylation is an essential step in ATP generation in O. formigenes, and the decarboxylase activity is stimulated by exogenous ADP. Despite the significant degree of structural conservation between the two homologous enzymes and the similarity in catalytic mechanism to other thiamin diphosphate-dependent enzymes, the active site residues of oxalyl-CoA decarboxylase are unique. A suggestion for the reaction mechanism of the enzyme is presented.     

The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal aclf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous additionof 10^?2 M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and its noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.     

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.     

The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes

Glucuronoxylans with a backbone of 1,4-linked β-D-xylosyl residues are ubiquitous in the secondary walls of gymnosperms and angiosperms. Xylans have been reported to be present in hornwort cell walls, but their structures have not been determined. In contrast, the presence of xylans in the cell walls of mosses and liverworts remains a subject of debate. Here we present data that unequivocally establishes that the cell walls of leafy tissue and axillary hair cells of the moss Physcomitrella patens contain a glucuronoxylan that is structurally similar to glucuronoxylans in the secondary cell walls of vascular plants. Some of the 1,4-linked β-D-xylopyranosyl residues in the backbone of this glucuronoxylan bear an α-D-glucosyluronic acid (GlcpA) sidechain at O-2. In contrast, the lycopodiophyte Selaginella kraussiana synthesizes a glucuronoxylan substituted with 4-O-Me-α-D-GlcpA sidechains, as do many hardwood species. The monilophyte Equisetum hyemale produces a glucuronoxylan with both 4-O-Me-α-D-GlcpA and α-D-GlcpA sidechains, as does Arabidopsis. The seedless plant glucuronoxylans contain no discernible amounts of the reducing-end sequence that is characteristic of gymnosperm and eudicot xylans. Phylogenetic studies showed that the P. patens genome contains genes with high sequence similarity to Arabidopsis CAZy family GT8, GT43 and GT47 glycosyltransferases that are likely involved in xylan synthesis. We conclude that mosses synthesize glucuronoxylan that is structurally similar to the glucuronoxylans present in the secondary cell walls of lycopodiophytes, monilophytes, and many seed-bearing plants, and that several of the glycosyltransferases required for glucuronoxylan synthesis evolved before the evolution of tracheophytes.     

Correlation between the ability of tumor cells to resist humoral immune attack and their ability to synthesize lipid.

Agents that increase (certain metabolic inhibitors, chemotherapeutic agents, and x-irradiation), decrease (hormones), or have no effect (hyperthermia) on the susceptibility of line-1 and line-10 guinea pig hepatoma cells to humoral immune attack were studied for their effects on the ability of these tumor cells to synthesize macromolecules. A correlation was found between the drug-induced increase in sensitivity of these cells to antibody-C mediated killing and the loss of their ability to incorporate fatty acids into complex cellular lipids. Similarly, the hormone-induced increase in resistance of the cells to killing was accompanied by an enhancement in complex lipid synthesis by these cells was also observed after the cells were exposed to physical means of insult (x-irradiation or hyperthermia). No correlation was found between the sensitivity of the cells to antibody-C mediated killing and their ability to synthesize DNA, RNA, protein, or complex carbohydrate, or their capacity for de novo lipid synthesis as measured by incorporation of acetate and glycerol into cellular macromolecules. The assembly of free fatty acids into complex lipid moieties is therefore proposed to be of fundamental importance for the ability of the tumor cells to resist humoral immune killing.     

The appearance of an ornithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures.

Quiescent, contact inhibited H-35 rat hepatoma cell cultures maintained in minimal essential medium contain a very low level of ornithine decarboxylase activity. However, 2 h after the addition of 10% fetal calf serum to the culture medium, the enzyme activity increases by approx. 100-fold. This increase can be completely inhibited by the simultaneous addition of 10(-2) M putrescine. The presence of putrescine elicits the appearance of an intracellular inhibitor of ornithine decarboxylase. This inhibitor of ornithine decarboxylase has a molecular weight of 26500, is sensitive to the action of chymotrypsin and is noncompetitive with respect to ornithine. The intracellular appearance of this inhibitor is sensitive to cycloheximide but is only partially inhibited by actinomycin D.     

Aspergillus oryzae NRRL 35191 from coffee, a non-toxigenic endophyte with the ability to synthesize kojic acid

Aspergillus oryzae NRRL 35191 was isolated as an endophyte from coffee leaves and found to produce kojic acid (KA) in culture. When inoculated into cacao seedlings (Theobroma cacao), A. oryzae grew endophytically and synthesized KA in planta. Cacao seedlings inoculated with A. oryzae produced higher levels of caffeine than non-inoculated ones. Aspergillus oryzae may be a useful endophyte to introduce to cacao since it grows non-pathogenically and induces the caffeine defense response that may make the plant more tolerant to insects and pathogens.     

A novel type of putrescine (diamine)-acetylating enzyme from the nematode Ascaris suum.

Component polypeptides of both the bovine lens and pituitary multicatalytic proteinase complexes demonstrate different immunoreactivities with a polyclonal antiserum raised against the purified pituitary enzyme. Four (Mr 24000, 26000, 34000 and 38000) of eight bands that have been resolved by SDS/polyacrylamide-gel electrophoresis are stained in immunoblot experiments. Monospecific antibodies obtained from this antiserum by affinity purification from the 38000- and 34000-Mr bands of the lens enzyme bound equally well to either band, but showed little or no binding to the 26000- and 24000-Mr bands upon immunoblotting. Antibody affinity-purified from the 24000-Mr band showed comparable binding to the 24000-, 34000- or 38000-Mr band. One explanation of these results is that the 24000-Mr polypeptide is derived from the higher-Mr polypeptide(s) and has lost some of the common immunodeterminants.