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PubMed2000

Sequence analysis and functional characterization of the violacein biosynthetic pathway from Chromobacterium violaceum.

Violacein is a purple-colored, broad-spectrum antibacterial pigment that has a dimeric structure composed of 5-hydroxyindole, oxindole and 2-pyyrolidone subunits formed by the condensation of two modified tryptophan molecules. The violacein biosynthetic gene cluster from Chromobacterium violaceum was characterized by DNA sequencing, transposon mutagenesis, and chemical analysis of the pathway intermediates produced heterologously in Escherichia. coli. The violacein biosynthetic gene cluster spans eight kilobases and is comprised of the four genes, vioABCD, that are necessary for violacein production. Sequence analysis suggests that the products of vioA, vioC and vioD are nucleotide-dependent monooxygenases. Disruption of vioA or vioB completely abrogates the biosynthesis of violacein intermediates, while disruption of the vioC or vioD genes results in the production of violacein precursors.

Amino Acid Sequence↗
PubMed2007

Dissecting the biosynthetic pathway for the bypass1 root-derived signal.

The Arabidopsis BYPASS1 (BPS1) gene is required for normal root and shoot development. In bps1 mutants, grafting and root excision experiments have shown that mutant roots produce a transmissible signal that is capable of arresting shoot development. In addition, we previously showed that growth of bps1 mutants on the carotenoid biosynthesis inhibitor fluridone resulted in partial rescue of both leaf and root defects. These observations suggest that a single mobile carotenoid-derived signal affects both root and shoot development. Here, we describe further characterization of the bps1 root-derived signal using genetic and biosynthetic inhibitor approaches. We characterized leaf and root development in double mutants that combined the bps1 mutant with mutants that have known defects in genes encoding carotenoid processing enzymes or defects in responses to carotenoid-derived abscisic acid. Our studies indicate that the mobile signal is neither abscisic acid nor the MAX-dependent hormone that regulates shoot branching, and that production of the signal does not require the activity of any single carotenoid cleavage dioxygenase. In addition, our studies with CPTA, a lycopene cyclase inhibitor, show that signal production requires synthesis of beta-carotene and its derivatives. Furthermore, we show a direct requirement for carotenoids as signal precursors, as the GUN plastid-to-nucleus signaling pathway is not required for phenotypic rescue. Together, our results suggest that bps1 roots produce a novel mobile carotenoid-derived signaling compound.

Abscisic Acid↗
PubMed2004

Contribution of different biosynthetic pathways to species selectivity of aminoglycerophospholipids assembled into mitochondrial membranes of the yeast Saccharomyces cerevisiae.

In the yeast Saccharomyces cerevisiae, three pathways lead to the formation of cellular phosphatidylethanolamine (PtdEtn), namely the mitochondrial conversion of phosphatidylserine (PtdSer) to PtdEtn catalyzed by phosphatidylserine decarboxylase 1 (Psd1p), the equivalent reaction catalyzed by phosphatidylserine decarboxylase 2 (Psd2p) in the Golgi, and the CDP-ethanolamine branch of the so-called Kennedy pathway which is located to the microsomal fraction. To investigate the contributions of these three pathways to the cellular pattern of PtdEtn species (fatty acid composition) we subjected lipids of wild-type and yeast mutant strains with distinct defects in the respective pathways to mass spectrometric analysis. We also analyzed species of PtdSer and phosphatidylcholine (PtdCho) of these strains because formation of the three aminoglycerophospholipids is linked through their biosynthetic route. We demonstrate that all three pathways involved in PtdEtn synthesis exhibit a preference for the formation of C34:2 and C32:2 species resulting in a high degree of unsaturation in total cellular PtdEtn. In PtdSer, the ratio of unsaturated to saturated fatty acids is much lower than in PtdEtn, suggesting a high species selectivity of PtdSer decarboxylases. Finally, PtdCho is characterized by its higher ratio of C16 to C18 fatty acids compared to PtdSer and PtdEtn. In contrast to biosynthetic steps, import of all three aminoglycerophospholipids into mitochondria of wild-type and mutant cells is not highly specific with respect to species transported. Thus, the species pattern of aminoglycerophospholipids in mitochondria is mainly the result of enzyme specificities, but not of translocation processes involved. Our results support a model that suggests equilibrium transport of aminoglycerophospholipids between mitochondria and microsomes based on membrane contact between the two compartments.

Biological Transport↗
PubMed1994

Fermentative production of tryptophan by a stable recombinant strain of Corynebacterium glutamicum with a modified serine-biosynthetic pathway.

Introduction of plasmid pKW99, which coexpresses the deregulated 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and tryptophan-biosynthetic enzymes, into tryptophan-producing Corynebacterium glutamicum KY10894 resulted in a marked increase (54%) in yield of tryptophan production (43 g/liter), but incurred two problems. One was a decline in sugar consumption at the late stage of fermentation, and the other the loss of the plasmid in the absence of selective pressure. The retarded sugar assimilation was found to be attributed to the death of cells that arose from the detrimental action of indole, the last intermediate in the tryptophan pathway, accumulated as a by-product. A chain of these events simultaneously disappeared when serine, the other substrate of the final reaction by tryptophan synthase, was added. These results indicated that a limiting supply of serine was the cause of the decline in the sugar consumption. Thus, to increase carbon flux into serine, the gene for 3-phosphoglycerate dehydrogenase (PGD), the first enzyme in the serine pathway, was cloned from wild-type C. glutamicum ATCC 31833 and joined onto pKW99 to generate pKW9901. Strain KY10894 transformed with pKW9901 favorably consumed sugar through fermentation with accumulating little indole. Furthermore, on the basis of the observation that serine in the medium was consumed rapidly by the recombinant cells, we developed a unique plasmid stabilization system composed of KY9218 (a PGD-deficient serine-requiring strain of KY10894) and pKW9901: In its combination, cells lacking the plasmid should not proliferate in the fermentation medium which does not contain serine. Even if selective pressure was not applied, the modified strain KY9218 with pKW9901 stably maintained the plasmid during fermentation and produced 50 g/liter of tryptophan in a 61% increased yield relative to strain KY10894.

Carbohydrate Dehydrogenases↗
PubMed2004

Biosynthetic pathways to isocyanides and isothiocyanates; precursor incorporation studies on terpene metabolites in the tropical marine sponges Amphimedon terpenensis and Axinyssa n.sp.

The biosynthetic origins of the isocyanide and isothiocyanate functional groups in the marine sponge metabolites diisocyanoadociane (1), 9-isocyanopupukeanane (10) and 9-isothiocyanatopupukeanane (11) are probed by the use of [(14)C]-labelled precursor experiments. Incubation of the sponge Amphimedon terpenensis with [(14)C]-labelled thiocyanate resulted in radioactive diisocyanoadociane (1) in which the radiolabel is specifically associated with the isocyanide carbons. As expected, cyanide and thiocyanate were confirmed as precursors to the pupukeananes 10 and 11 in the sponge Axinyssa n.sp.; additionally these precursors labelled 2-thiocyanatoneopupukeanane (12) in this sponge. To probe whether isocyanide-isothiocyanate interconversions take place at the secondary metabolite level, the advanced precursor bisisothiocyanate 17 was supplied to A. terpenensis, but did not result in significant labelling in the natural product isocyanide 1. In contrast, in the sponge Axinyssa n.sp., feeding of [(14)C]-9-isocyanopupukeanane (10) resulted in isolation of radiolabelled 9-isothiocyanatopupukeanane 11, while the feeding of [(14)C]-11 resulted in labelled isocyanide 10. These results show conclusively that isocyanides and isothiocyanates are interconverted in the sponge Axinyssa n.sp., and confirm the central role that thiocyanate occupies in the terpene metabolism of this sponge.

Animals↗
PubMed1999

Isolation and characterization of experimentally induced, aflatoxin biosynthetic pathway deletion mutants of Aspergillus parasiticus.

A plasmid vector (pDEL2) was engineered for the purpose of introducing a deletion within the aflatoxin (AF) biosynthetic gene cluster of Aspergillus parasiticus. The vector was constructed by PCR amplification of a region of the AF gene cluster from an A. parasiticus isolate that had undergone an aberrant recombinational event during transformation with a nor A-niaD gene disruption vector. This recombinational event resulted in the deletion of an approximately 6-kb region of the AF gene cluster and accumulation of the AF precursor averantin (AVN). Northern hybridization analysis confirmed that the deletion event resulted in no detectable transcription of the norA gene or the AF biosynthetic genes, avnA, verA, and ver-1. Transformation of A. parasiticus RHN1 with pDEL2 resulted in 16% of the transformants accumulating AVN. Southern hybridization analysis of randomly selected AVN-accumulating transformants indicated that all had undergone a double-crossover homologous, recombinational event resulting in the 6-kb norA to avnA deletion within the AF gene cluster. Aflatoxin precursor feeding studies performed on one of the AVN-accumulating, RHN1(pDEL2) transformants confirmed that the enzyme activities associated with the deleted genes were no longer present.

Aflatoxins↗
PubMed2003

Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma).

The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.

Alkyl and Aryl Transferases↗
PubMed1994

Genetic analysis of the acetan biosynthetic pathway in Acetobacter xylinum.

We have identified, cloned and sequenced an 8422 base pair fragment of Acetobacter xylinum genomic DNA containing part of the acetan biosynthetic gene cluster. Computer analysis of the nucleotide sequence data generated revealed the presence of six open reading frames. Comparison of the translated sequences of putative genes to the amino acid sequences of genes from other organisms was used to assign functions to the aceA, aceC and manB genes. These genes were predicted to encode a UDP-glycosyl transferase, a GDP-mannosyl transferase and a phosphomannose isomerase/GDP-mannose pyrophosphorylase, respectively.

Bacterial Proteins↗
PubMed1993

A bifunctional protein in the folate biosynthetic pathway of Streptococcus pneumoniae with dihydroneopterin aldolase and hydroxymethyldihydropterin pyrophosphokinase activities.

A protein encoded by sulD, one of four genes in a previously cloned folate biosynthetic operon of Streptococcus pneumoniae, had been shown to harbor 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase activity. This SulD protein was purified and shown now to harbor also dihydroneopterin aldolase activity. The bifunctional protein therefore catalyzes two successive steps in folate biosynthesis. The aldolase activity can be ascribed to the N-terminal domain of the SulD polypeptide, and the pyrophosphokinase activity can be ascribed to the C-terminal domain. Homologs of the dihydroneopterin aldolase domain were identified in other species, in one of which the domain was encoded as a separate polypeptide. The native SulD protein is a trimer or tetramer of a 31-kDa subunit, and it dissociated reversibly after purification. Dihydroneopterin aldolase activity required the multimeric protein, whereas pyrophosphokinase was expressed by the monomeric form. With purified SulD, the amount of 6-hydroxymethyl-7,8-dihydropterin product formed by the aldolase was proportional to the fourth power of the enzyme concentration, as expected for a reversibly dissociating tetramer. By identifying the gene encoding dihydroneopterin aldolase, this work extends our understanding of the molecular basis of the folate biosynthetic system common to many organisms.

Aldehyde-Lyases↗
PubMed1972

Separation of mitochondrial membranes of Neurospora crassa. II. Submitochondrial localization of the isoleucine-valine biosynthetic pathway.

Separation of Neurospora mitochondrial outer membranes from the inner membrane/matrix fraction was effected by digitonin treatment and discontinuous density gradient centrifugation. The solubilization of four isoleucine-valine biosynthetic enzymes was studied as a function of digitonin concentration and time of incubation in the detergent. The kinetics of the appearance of valine biosynthetic function in fractions outside of the inner membrane/matrix fraction, coupled with enzyme solubilization patterns similar to that for the matrix marker, mitochondrial malate dehydrogenase, indicate that the four isoleucine-valine pathway enzymes are localized in the mitochondrial matrix.

Alcohol Oxidoreductases↗
PubMed2006

Cytosolic triacylglycerol biosynthetic pathway in oilseeds. Molecular cloning and expression of peanut cytosolic diacylglycerol acyltransferase.

Triacylglycerols (TAGs) are the most important storage form of energy for eukaryotic cells. TAG biosynthetic activity was identified in the cytosolic fraction of developing peanut (Arachis hypogaea) cotyledons. This activity was NaF insensitive and acyl-coenzyme A (CoA) dependent. Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the final step in TAG biosynthesis that acylates diacylglycerol to TAG. Soluble DGAT was identified from immature peanuts and purified by conventional column chromatographic procedures. The enzyme has a molecular mass of 41 +/- 1.0 kD. Based on the partial peptide sequence, a degenerate probe was used to obtain the full-length cDNA. The isolated gene shared less than 10% identity with the previously identified DGAT1 and 2 families, but has 13% identity with the bacterial bifunctional wax ester/DGAT. To differentiate the unrelated families, we designate the peanut gene as AhDGAT. Expression of peanut cDNA in Escherichia coli resulted in the formation of labeled TAG and wax ester from [14C]acetate. The recombinant E. coli showed high levels of DGAT activity but no wax ester synthase activity. TAGs were localized in transformed cells with Nile blue A and oil red O staining. The recombinant and native DGAT was specific for 1,2-diacylglycerol and did not utilize hexadecanol, glycerol-3-phosphate, monoacylglycerol, lysophosphatidic acid, and lysophosphatidylcholine. Oleoyl-CoA was the preferred acyl donor as compared to palmitoyl- and stearoyl-CoAs. These data suggest that the cytosol is one of the sites for TAG biosynthesis in oilseeds. The identified pathway may present opportunities of bioengineering oil-yielding plants for increased oil production.

Amino Acid Sequence↗
PubMed1998

Differential involvement of indole-3-acetic acid biosynthetic pathways in pathogenicity and epiphytic fitness of Erwinia herbicola pv. gypsophilae.

Erwinia herbicola pv. gypsophilae (Ehg), which induces galls on Gypsophila paniculata, harbors two major pathways for indole-3-acetic acid (IAA) synthesis, the indole-3-acetamide (IAM) and indole-3-pyruvate (IPyA) routes, as well as cytokinin biosynthetic genes. Mutants were generated in which the various biosynthetic routes were disrupted separately or jointly in order to assess the contribution of IAA of various origins and cytokinins to pathogenicity and epiphytic fitness. Inactivation of the IAM pathway or cytokinin biosynthesis caused the largest reduction in gall size. Inactivation of the IPyA pathway caused a minor, nonsignificant decrease in pathogenicity. No further reduction in gall size was observed by the simultaneous inactivation of both IAA pathways only or in combination with that of cytokinin production. However, inactivation of the IPyA pathway caused a 14-fold reduction in the population of Ehg on bean plants. Inactivation of the IAM pathway or cytokinin production did not affect epiphytic fitness. While the apparent transcriptional activity of iaaM-inaZ fusion increased slightly in cells of Ehg on bean and gypsophila leaves, compared with that in culture, very high levels of induction were observed in cells injected into gypsophila stems. In contrast, moderate levels of induction of ipdC-inaZ in Ehg were observed on leaves of these plants and in gypsophila stems, when compared with that in culture. These results suggest that the IAM pathway is involved primarily in gall formation and support the main contribution of the IpyA pathway to the epiphytic fitness of this bacterial species.

Base Sequence↗
PubMed2005

TDP-mycaminose biosynthetic pathway revised and conversion of desosamine pathway to mycaminose pathway with one gene.

Analysis of the tylosin gene cluster in Streptomyces fradiae uncovered an ORF, tyl1a, homologous to a hexose 3,4-isomerase found in Aneurinibacillus thermoaerophilus. Inclusion of the tyl1a gene along with other mycaminose biosynthetic genes (tylB, tylM1, tylM2, tylM3) identified in previous studies in an in vivo expression system successfully reconstituted the mycaminose pathway. Expression of tyl1a alone in the S venezuelae KdesI mutant converted a desosamine pathway to a mycaminose pathway. These results strongly support the role of Tyl1a as a TDP-4-keto-6-deoxy-d-glucose 3,4-isomerase.

Anti-Bacterial Agents↗
PubMed2006

Trafficking in persulfides: delivering sulfur in biosynthetic pathways.

The presence of sulfur in cofactors has been appreciated for over a century, but the trafficking and delivery of sulfur to cofactors and nucleosides is still not fully understood. In the last decade, great strides have been made toward understanding those processes and the enzymes that conduct them, including cysteine desulfurases and rhodanese homology domain proteins. The persulfide group (R-S-SH) predominantly serves as the sulfur donor, and sulfur incorporation pathways share enzymes to a remarkable degree. Mechanisms for the use of persulfide groups are illustrated with the relatively simple case of 4-thiourdine generation, and further possibilities are illuminated by the 2-thiouridine and cofactor biosynthetic systems. The rationale and ramifications of sharing enzymes between sulfur incorporation pathways are discussed, including implications for interpreting genetic or genomic data that indicate a role for a sulfur transfer protein in a particular biological process.

Alanine↗
PubMed1999

Functional coupling between various phospholipase A2s and cyclooxygenases in immediate and delayed prostanoid biosynthetic pathways.

Several distinct phospholipase A2s (PLA2s) and two cyclooxygenases (COXs) were transfected, alone or in combination, into human embryonic kidney 293 cells, and their functional coupling during immediate and delayed prostaglandin (PG)-biosynthetic responses was reconstituted. Signaling PLA2s, i.e. cytosolic PLA2 (cPLA2) (type IV) and two secretory PLA2s (sPLA2), types IIA (sPLA2-IIA) and V (sPLA2-V), promoted arachidonic acid (AA) release from their respective transfectants after stimulation with calcium ionophore or, when bradykinin receptor was cotransfected, with bradykinin, which evoked the immediate response, and interleukin-1 plus serum, which induced the delayed response. Experiments on cells transfected with either COX alone revealed subtle differences between the PG-biosynthetic properties of the two isozymes in that COX-1 and COX-2 were favored over the other in the presence of high and low exogenous AA concentrations, respectively. Moreover, COX-2, but not COX-1, could turn on endogenous AA release, which was inhibited by a cPLA2 inhibitor. When PLA2 and COX were coexpressed, AA released by cPLA2, sPLA2-IIA and sPLA2-V was converted to PGE2 by both COX-1 and COX-2 during the immediate response and predominantly by COX-2 during the delayed response. Ca2+-independent PLA2 (iPLA2) (type VI), which plays a crucial role in phospholipid remodeling, failed to couple with COX-2 during the delayed response, whereas it was linked to ionophore-induced immediate PGE2 generation via COX-1 in marked preference to COX-2. Finally, coculture of PLA2 and COX transfectants revealed that extracellular sPLA2s-IIA and -V, but neither intracellular cPLA2 nor iPLA2, augmented PGE2 generation by neighboring COX-expressing cells, implying that the heparin-binding sPLA2s play a particular role as paracrine amplifiers of the PG-biosynthetic response signal from one cell to another.

Animals↗
PubMed2001

Effects of aflastatin A, an inhibitor of aflatoxin production, on aflatoxin biosynthetic pathway and glucose metabolism in Aspergillus parasiticus.

Aflastatin A inhibits aflatoxin production by Aspergillus parasiticus via an unknown mechanism. We found that aflastatin A clearly inhibited production of norsolorinic acid, an early biosynthetic intermediate of aflatoxin, at a concentration of 0.25 microg/ml. Reverse-transcriptase polymerase chain reaction (RT-PCR), and real-time quantitative PCR (TaqMan PCR) experiments indicated that the transcription of genes encoding aflatoxin biosynthetic enzymes (pksA, ver-1, and omtA) and a gene encoding a regulatory protein for expression of the biosynthetic enzymes (aflR) were significantly reduced by the addition of aflastatin A. We also found that aflastatin A elevated the glucose consumption and ethanol accumulation by A. parasiticus, and repressed transcription of genes involved in ethanol utilization. These results suggest that aflastatin A inhibits a very early step in aflatoxin biosynthesis prior to the transcription of aflR and can influence glucose metabolism in the fungus.

Aflatoxins↗
PubMed1999

Functional association of type IIA secretory phospholipase A(2) with the glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan in the cyclooxygenase-2-mediated delayed prostanoid-biosynthetic pathway.

An emerging body of evidence suggests that type IIA secretory phospholipase A(2) (sPLA(2)-IIA) participates in the amplification of the stimulus-induced cyclooxygenase (COX)-2-dependent delayed prostaglandin (PG)-biosynthetic response in several cell types. However, the biological importance of the ability of sPLA(2)-IIA to bind to heparan sulfate proteoglycan (HSPG) on cell surfaces has remained controversial. Here we show that glypican, a glycosylphosphatidylinositol (GPI)-anchored HSPG, acts as a physical and functional adaptor for sPLA(2)-IIA. sPLA(2)-IIA-dependent PGE(2) generation by interleukin-1-stimulated cells was markedly attenuated by treatment of the cells with heparin, heparinase or GPI-specific phospholipase C, which solubilized the cell surface-associated sPLA(2)-IIA. Overexpression of glypican-1 increased the association of sPLA(2)-IIA with the cell membrane, and glypican-1 was coimmunoprecipitated by the antibody against sPLA(2)-IIA. Glypican-1 overexpression led to marked augmentation of sPLA(2)-IIA-mediated arachidonic acid release, PGE(2) generation, and COX-2 induction in interleukin-1-stimulated cells, particularly when the sPLA(2)-IIA expression level was suboptimal. Immunofluorescent microscopic analyses of cytokine-stimulated cells revealed that sPLA(2)-IIA was present in the caveolae, a microdomain in which GPI-anchored proteins reside, and also appeared in the perinuclear area in proximity to COX-2. We therefore propose that a GPI-anchored HSPG glypican facilitates the trafficking of sPLA(2)-IIA into particular subcellular compartments, and arachidonic acid thus released from the compartments may link efficiently to the downstream COX-2-mediated PG biosynthesis.

Cell Line↗