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PubMed2004

The importance of a functional trehalose biosynthetic pathway for the life of yeasts and fungi.

The view of the role of trehalose in yeast has changed in the last few years. For a long time considered a reserve carbohydrate, it gained new importance when its function in the acquisition of thermotolerance was demonstrated. More recently the cellular processes in which the trehalose biosynthetic pathway has been implicated range from the control of glycolysis to sporulation and infectivity by certain fungal pathogens. There is now enough experimental evidence to conclude that trehalose 6-phosphate, an intermediate of trehalose biosynthesis, is an important metabolic regulator in such different organisms as yeasts or plants. Its inhibition of hexokinase plays a key role in the control of the glycolytic flux in Saccharomyces cerevisiae but other, likely important, sites of action are still unknown. We present examples of the phenotypes produced by mutations in the two steps of the trehalose biosynthetic pathway in different yeasts and fungi, and whenever possible examine the molecular explanations advanced to interpret them.

Fungi↗

PubMed1994

The hexosamine biosynthetic pathway and glucose-induced down regulation of glucose transport in L6 myotubes.

Based on experiments in cultured adipocytes, it has been proposed that glucose-induced down regulation of glucose transport is mediated by the conversion of fructose-6-phosphate to glucosamine-6-phosphate via the first and rate-determining enzyme of the hexosamine biosynthetic pathway, glutamine: fructose-6-phosphate amidotransferase (glutamine hexosephosphate aminotransferase). Evidence for this assertion was: (a) L-glutamine, the provider group for the aminotransferase was essential; (b) two inhibitors of glutamine hexosephosphate aminotransferase, 6-diazo-5-oxonorleucine (L form) and azaserine, blocked glucose-induced down regulation of glucose transport; (c) azaserine inhibited the activity of the aminotransferase, (d) glucosamine, which enters the hexosamine pathway distal to this enzyme was 40-times more potent than glucose; and (e) azaserine was unable to block the effect of glucosamine. Since muscle is quantitatively much more important than adipose tissue for whole body glucose utilization, we sought to determine if the hexosamine pathway was involved in glucose-induced down regulation of glucose transport in L6 myotubes. Glucose was effective, both in the presence and absence of glutamine in the incubation media. Glucosamine was also effective but was as equipotent as glucose. Small amounts of glutamine hexosephosphate aminotransferase were present in the L6 myotubes and although the leucine derivative (20 microM) inhibited the enzyme, it did not impair glucose-induced down regulation of glucose transport. Total GLUT-1 levels were similar when the cells were incubated in the absence or presence of 5 mM glucose or glucosamine although glucosamine was associated with a marked increase in a lower molecular weight band. These results do not suggest that the hexosamine biosynthetic pathway is involved in glucose-induced down regulation of glucose transport in L6 myotubes. Thus, this phenomenon is regulated differently in muscle and fat.

Animals↗

PubMed2004

Expression of terpenoid indole alkaloid biosynthetic pathway genes corresponds to accumulation of related alkaloids in Catharanthus roseus (L.) G. Don.

Madagascar periwinkle, Catharanthus roseus (L.) G. Don, a medicinally important plant, produces anticancer dimeric alkaloids, vinblastine and vincristine, in the leaves and accumulates antihypertensive alkaloids, ajmalicine and serpentine, in the roots. This plant grows wild in distant tropical and sub-tropical geographical locations with different agro-climates and shows wide variations in morphological and alkaloid yield-related traits. In order to understand the correlation between the expression of terpenoid indole alkaloid (TIA) pathway genes and accumulation of related alkaloids, six different genetic resources of C. roseus, including the medicinal cultivars Nirmal, Prabal, Dhawal, the mutants gsr-3 and gsr-6, and one horticultural variety, Pacifica blush, were studied. The expression profiles of one early and two late TIA biosynthetic pathway genes, namely, strictosidine synthase, desacetoxyvindoline 4-hydroxylase and deacetyl vindoline 4-O-acetyl transferase were analyzed in these plants. A positive correlation between transcript abundance and accumulation of related alkaloids was observed in the different genetic resources. The potential of these TIA biosynthetic pathway genes for use in screening of high-yielding C. roseus germplasm has been discussed.

Acetyltransferases↗

PubMed1994

Targeting of the polyhydroxybutyrate biosynthetic pathway to the plastids of Arabidopsis thaliana results in high levels of polymer accumulation.

In the bacterium Alcaligenes eutrophus, three genes encode the enzymes necessary to catalyze the synthesis of poly[(R)-(-)-3-hydroxybutyrate] (PHB) from acetyl-CoA. In order to target these enzymes into the plastids of higher plants, the genes were modified by addition of DNA fragments encoding a pea chloroplast transit peptide, a constitutive plant promoter, and a poly(A) addition sequence. Each of the modified bacterial genes was introduced into Arabidopsis thaliana by Agrobacterium-mediated transformation, and plants containing all three genes were obtained by sexual crosses. These plants accumulated PHB up to 14% of the dry weight as 0.2- to 0.7-micron granules within plastids. In contrast to earlier experiments in which expression of the PHB biosynthetic pathway in the cytoplasm led to a deleterious effect on growth, expression of the PHB biosynthetic pathway in plastids had no obvious effect on the growth or fertility of the transgenic plants and resulted in a 100-fold increase in the amount of PHB that accumulated. We conclude that there does not appear to be any biological barrier to high-level production of PHB in higher plants. The high level of PHB accumulation also suggests that the synthesis of plastid acetyl-CoA is regulated by a mechanism which responds to metabolic demand.

Acyltransferases↗

PubMed1990

Rhodobacter capsulatus genes involved in early steps of the bacteriochlorophyll biosynthetic pathway.

Three open reading frames in the Rhodobacter capsulatus photosynthesis gene cluster, designated F0, F108, and F1025, were disrupted by site-directed mutagenesis. Mutants bearing insertions in these reading frames were defective in converting protoporphyrin IX to magnesium-protoporphyrin monomethyl ester, protochlorophyllide to chlorophyllide a, and magnesium-protoporphyrin monomethyl ester to protochlorophyllide, respectively. These results demonstrate that the genes examined most likely encode enzyme subunits that catalyze steps common to plant and bacterial tetrapyrrole photopigment biosynthetic pathways. The open reading frames were found to be part of a large 11-kilobase operon that encodes numerous genes involved in early steps of the bacteriochlorophyll a biosynthetic pathway.

Amino Acid Sequence↗

PubMed1978

The biosynthetic pathway of new polyamines in Caldariella acidophila.

1. Spermidine and sym-nor-spermine (1,11-diamino-4,8-diazaundecane) were identified as the major components of the polyamine pool in Caldariella acidophila, an extreme thermoacidophilic bacterium. A minor component, a new polyamine, sym-nor-spermidine (1,7-diamino-4-azaheptane) was isolated and characterized. 2. To elucidate the biosynthetic pathway, labelled methionine, putrescine, spermidine and spermine were fed to Caldariella acidophila. Incubation of the bacterium in the presence of putrescine or spermidine labelled in the tetramethylene moiety gave unlabelled sym-nor-spermidine and sym-nor-spermine, whereas the radioactivity of propylamine-labelled methionine or spermidine was incorporated into these molecules. No radioactivity was recovered in the polyamines pool when spermine was fed to Caldariella acidophila. 3. S-Adenosylmethionine and S-(5'-adenosyl)-3-methylthiopropylamine were identified as intermediates of the biosynthetic pathway; the cellular contents of the two sulphonium compounds, measured with a new isotope-dilution technique, are 60 and 15nmol/g wet wt. of cells respectively. 4. The above results are indicative of a new pathway characterized by three propylamine-transfer reactions, decarboxylated S-adenosylmethionine being the common donor of the propylamine moiety. The reactions yielding sym-nor-spermidine and sym-nor-spermine are reported for the first time. 5. The probable intermediates related to the catabolism of the tetramethylene moiety of spermidine, gamma-aminobutyraldehyde, gamma-aminobutyric acid or Delta(1)-pyrroline were not detectable. Experiments with [3-aminopropyl-3(n)-(3)H]spermidine trihydrochloride plus [tetramethylene-1,4-(14)C]spermidine trihydrochloride gave rise to an amount of labelled CO(2) equivalent to the spermidine catabolized.

Bacteria↗

PubMed2005

Functional characterization of dehydratase/aminotransferase pairs from Helicobacter and Campylobacter: enzymes distinguishing the pseudaminic acid and bacillosamine biosynthetic pathways.

Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-alpha-D-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His(6)-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-beta-L-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-beta-L-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-alpha-D-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-alpha-D-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-alpha-D-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-alpha-D-glucopyranose.

Campylobacter jejuni↗

PubMed2002

Role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in symbiosis.

Rhizobia require the availability of 20 amino acids for the establishment of effective symbiosis with legumes. Some of these amino acids are synthesized by rhizobium, whereas the remaining are supplied by the host plant. The supply from plant appears to be plant-type specific. Alfalfa provides arginine, cysteine, isoleucine, valine and tryptophan, and cowpea and soybean provide histidine. The production of ornithine and anthranilic acid, the intermediates in the biosynthetic pathways of arginine and tryptophan, respectively, seems to be essential for effective symbiosis of Sinorhizobium meliloti with alfalfa. The expression of ilvC gene of S. meliloti is required for induction of nodules on the roots of alfalfa plants. An undiminished metabolic flow through the rhizobial pathways for the synthesis of purines and pyrimidines and the synthesis of biotin, nicotinic acid, riboflavin and thiamine by rhizobium appear to be requirements for normal symbiosis. To the best of our knowledge, this is the first review article on the role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in rhizobium-legume symbiosis. The scientific developments of about 35 years in this field have been reviewed.

Amino Acids↗

PubMed2000

[Research of indole-3-acetic acid biosynthetic pathway of Klebsiella oxytoca SG-11 by HPLC and GC-MS].

The plant growth promoting bacteria are closely associated to plant. The bacteria are used to adhering to plant rhizoplane, promoting plant growth by fixing nitrogen from atmosphere, secreting stimulating substances or producing antagonistic to plant pathogens. It was indicated that the biological nitrogen fixation played an important role in plant growth promoting function. In fact, it was verified recently by overall research that IAA does it. Therefore research of IAA production and biosynthetic pathway of plant growth-promoting bacteria is much more important. The various ways of IAA production indicated the strong or weak promoting function of bacterium to plants in general. The purpose of this paper is to determine whether IAA exists in cultured medium of Klebsiella axytoca SG-11 and biosynthetic pathway of IAA, in order to opimize cultural conditions for IAA production. Klebsiella axytoca SG-11 is a plant growth promoting bacterium, isolated from rice rhizoplane, which can fix nitrogen. The supernatant of SG-11 cultured medium determined by HPLC showed that 47.4 mg/L of IAA existed in LB medium and 1.2 mg/L of IAA, in basal medium. IAA in metabolite was identified by GC/MS as well. The intermediate determination of tryptamine, indole-3-acetamide, tryptophol and indole-3-acetonitrile indirectly indicated that IAA was biosynthesized in a pathway of indole-3-pyruvic acid. Meanwhile, tryptophol in metabolite of SG-11 was verified by GC/MS. The direct intermediates of indole-3-pyruvic acid and indole-3-acetaldehyde in the pathway can not be determined, because both are unstable under normal condition. As reversible conversion existed between indole-3 pyruvic aldehyde and tryptophol, the presence of tryptophol also proved the pathway of indole-3-pyruvic acid in the synthesis of IAA by Klebsiella axytoca SG-11. The results laid basis for further research of plant growth-promoting function of the bacterium.

Chromatography, High Pressure Liquid↗

PubMed2005

A cascade analysis of the interaction of mercury and coproporphyrinogen oxidase (CPOX) polymorphism on the heme biosynthetic pathway and porphyrin production.

Mercury (Hg) exposure in various forms remains a persistent public health concern in many parts of the world. In previous studies, we have described a biomarker of mercury exposure characterized by increased urinary concentrations of specific porphyrins, pentacarboxyporphyrin (5-CP) and coproporphyrin (4-CP), and the atypical keto-isocoproporphyrin (KICP), based on selective interference with the fifth (uroporphyrinogen decarboxylase, UROD) and sixth (coproporphyrinogen oxidase, CPOX) enzymes of the heme biosynthetic pathway. Whereas this response occurs in a predictable manner among approximately 85% of subjects with Hg exposure, an atypical porphyrinogenic response (APR) has been observed in approximately 15% of Hg-exposed persons, in which the three porphyrins that are affected by Hg, i.e., 5-CP, 4-CP and, KICP, are excreted in substantial excess of that predicted on the basis of Hg exposure alone. This APR has been attributed to a specific polymorphism in exon 4 of the CPOX gene (CPOX4). In the present study, we sought to further confirm the hypothesis that the observed changes in porphyrin excretion patterns might serve as a biomarker of Hg exposure and potential toxicity by statistically modeling the cascading effects on porphyrin concentrations within the heme biosynthetic pathway of Hg exposure and CPOX4 polymorphism in a human population with long-term occupational exposure to elemental mercury. Our results are highly consistent with this hypothesis. After controlling for precursor porphyrin concentrations, we demonstrated that 5-CP and 4-CP are independently associated with Hg concentration, while KICP is associated only with the CPOX4. An unpredicted association of Hg with heptacarboxyporphyrin (7-CP) may indicate a previously unidentified point of mercury inhibition of UROD. These findings lend further support to the proposed utility of urinary porphyrin changes as a biomarker of exposure and potential toxicity in subjects with mercury exposure. Additionally, these findings demonstrate the successful application of a computational model for characterizing complex metabolic responses and interactions associated with both toxicant exposure and genetic variation in human subjects.

Adult↗

PubMed1988

Biosynthetic pathways for the Leb and Y glycolipids in the gastric carcinoma cell line KATO III as analyzed by a novel assay.

The biosynthetic pathways for the difucosylated type 1 and 2 glycolipids, Leb and Y, respectively, were investigated in the gastric carcinoma cell line KATO III, using a novel chromatogram binding assay. The type of fucosylation obtained was deduced from the binding pattern of monoclonal antibodies specific for the biosynthesized glycolipid products using microsomal fractions as the source of enzyme, pure glycolipids and non-radioactive GDP-fucose as acceptor and donor substrates, respectively. The Leb glycolipid (Fuc alpha 1----2Gal beta 1----3GlcNAc(4----1 alpha Fuc) beta 1----3LacCer) was synthesized mainly via the blood group H, type 1, precursor (Fuc alpha 1----2Gal beta 1----3GlcNAc beta 1----3LacCer). However, the Lea glycolipid (Gal beta 1----3GlcNAc(4----1 alpha Fuc)beta 1----3LacCer) also served as a precursor for the alpha 1----2 fucosyltransferase, thus allowing conversion of Lea to Leb. This biosynthetic route represents either an "aberrant" specificity of the Fuc alpha 1----2 transferase associated with these gastric carcinoma cells and/or a new member of the alpha 1----2 fucosyltransferase family. The Y glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) was synthesized exclusively via the classical pathway using the blood group H type 2 glycolipid (Fuc alpha 1----2Gal beta 1----4GlcNAc beta 1----3LacCer) as precursor. The X glycolipid (Gal beta 1----4GlcNAc(3----1 alpha Fuc)beta 1----3LacCer) did not serve as an acceptor substrate for the alpha 1----2 fucosyltransferase(s) present. The use of non-radioactive sugar-nucleotides as donor substrate, defined glycolipid precursors as acceptor substrates and of specific monoclonal anti-glycolipid antibodies for detection provides a rapid and highly specific assay for analyzing biosynthetic pathways of glycosyltransferases.

Antigens↗

PubMed1962

Biosynthesis and biosynthetic pathways of pentoses in Escherichia coli.

Sable, Henry Z. (Western Reserve University, Cleveland, Ohio) and Elayne E. Cassisi. Biosynthesis and biosynthetic pathways of pentoses in Escherichia coli. J. Bacteriol. 84:1169-1172. 1962.-Resting glucose-adapted Escherichia coli supplied with glucose continues to synthesize pentose by the oxidative pathway characteristic of logarithmically growing glucose-adapted cells. This behavior is unlike that of acetate-adapted resting E. coli supplied with glucose, which continues to synthesize most of its pentose by the nonoxidative pathway characteristic of acetate-adapted cells. When infected with bacteriophage T2H, E. coli continues to use the oxidative pathway predominantly. This finding is in contrast to reports that infection with T6r+ bacteriophage increases the participation of a nonoxidative pathway. Resting glucose-adapted E. coli supplied with acetate-1-C(14) as sole carbon source synthesizes pentose by a pathway or pathways which cannot be assessed completely by methods previously developed (which are based on the relative labeling of C-1, C-2, and C-3 of the pentose) but which is most probably predominantly nonoxidative.

Acetates↗

PubMed1999

Enhanced uptake of [3H] spermidine by 9L rat brain tumors after direct intratumoral infusion of inhibitors of enzymes of the polyamine biosynthetic pathway.

We have been exploring the feasibility of delivering ionizing radiation to brain tumor cells by using tritium labeled polyamines. Polyamines are taken up preferentially by dividing cells and form noncovalent bonds with DNA. Their uptake can be enhanced by drugs which deplete endogenous polyamines. To test this in vivo, 9L cells were implanted in the striatal region of the brain in male Fisher 344 rats. Osmotic pumps containing trace amounts of [3H] spermidine or [3H] putrescine with either difluoromethylornithine or combinations of 3 inhibitors of enzymes of the polyamine biosynthetic pathway were implanted subcutaneously and were connected to intratumoral cannulas. After 14-16 days the brains were removed and sliced in the coronal plane. The diameters of the tumors were measured and tumor tissue was dissected from each slice, weighed and lysed for scintillation counting. It was found that difluoromethylornithine enhanced the uptake of [3H] putrescine while a combination of inhibitors of enzymes of the polyamine biosynthetic pathway enhanced the uptake of [3H] putrescine and [3H] spermidine producing a localized region of radioactivity in the 9L tumor. It is estimated that if the [3H] polyamines were at higher specific activity (commercially available), instead of the trace dose given here, the [3H] polyamine uptake would be sufficient to kill 9L tumor cells within a 2 to 3 week period.

Adenosylmethionine Decarboxylase↗

PubMed1995

Canine hereditary ceroid-lipofuscinosis: evidence for a defect in the carnitine biosynthetic pathway.

The ceroid-lipofuscinoses are a group of autosomal-recessive hereditary lysosomal storage diseases that have been characterized in humans and other mammalian species. In a canine model for the juvenile form of the human disease, a major constituent of the storage bodies is the subunit c protein of mitochondrial ATP synthase that contains an epsilon-N-trimethyllysine (TML) residue. TML is a precursor in carnitine biosynthesis. To determine whether accumulation of the TML-containing protein could result from a defect in the carnitine biosynthetic pathway, plasma carnitine and trimethyllysine levels were measured in homozygous affected, heterozygous carriers, and in normal dogs. When compared top normal animals, mean carnitine levels were reduced by 67% in affected and 50% in carrier dogs. Mean plasma TML levels were elevated almost 50% above control levels in the carriers, but were decreased by approximately 25% in the affected animals. The changes in plasma carnitine and TML levels in the carriers are consistent with the possibility that the disease involves a defect in the carnitine biosynthetic pathway. Secondary effects of the disease process may account for the apparently contradictory decrease in plasma TML levels in affected animals.

Animals↗

PubMed1996

Cloning and characterization of a maize cDNA encoding phytoene desaturase, an enzyme of the carotenoid biosynthetic pathway.

To study regulation of the plastid-localized maize carotenoid biosynthetic pathway, a cDNA encoding phytoene desaturase (PDS) was isolated and characterized. The DNA sequence of the maize Pds cDNA was determined and compared with available dicot Pds genes. The deduced PDS protein, estimated at 64.1 kDa (unprocessed), had a dinucleotide binding domain and conserved regions characteristic of other carotene desaturases. Alignment of available PDS sequences from distantly related organisms suggests that Pds has potential as a phylogenetic tool. By use of heterologous complementation in Escherichia coli, maize PDS was shown to catalyze two desaturation steps converting phytoene to zeta-carotene. RFLP (restriction fragment length polymorphism) mapping was used to place Pds on chromosome 1S near viviparous5 (vp5), and RT-PCR (reverse-transcriptase polymerase chain reaction) analysis indicated reduced Pds transcript in vp5 mutant relative to normal endosperm. Other phytoene-accumulating mutant endosperms, vp2 and white3 (w3), showed no difference in Pds transcript accumulation as compared with normal endosperm counterparts. RT-PCR analysis of Pds transcript accumulation in developing endosperm showed Pds was constitutively expressed. Therefore, endosperm carotenogensis is not regulated by increasing the level of Pds transcripts.

Amino Acid Sequence↗

PubMed1983

Enzymes of the de novo pyrimidine biosynthetic pathway in Toxoplasma gondii.

All six enzymes of the de novo biosynthetic pathway leading to the biosynthesis of UMP have been characterized in Toxoplasma gondii. The first three enzymes of the pathway, carbamyl phosphate synthetase-II (CPS-II), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) could be consistently separated by sucrose gradient centrifugation. Their molecular weights were estimated to be approximately 540 000, 140 000 and 70 000, respectively. The last two enzymes, orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase), cosedimented at the same position, corresponding also to a molecular weight of approximately 70 000. The fourth enzyme, dihydroorotate dehydrogenase (DHO-DHase), was associated with the particulate fraction. Apparent Km values for the respective enzymes were: CPS-II, MgATP2- (19.7 1.2 mM), L-glutamine (12.0 +/- 1.7 microM), ammonia (15.5 +/- 2.7 mM); ATCase, carbamyl phosphate (26.2 +/- 3.5 microM), L-aspartate (17.6 +/- 8.5 mM); DHOase (reverse direction) dihydroorotate (1.6 +/- 0.08 microM); ODCase, orotidine 5'-monophosphate (0.41 +/- 0.04 microM). MgUTP2- was found to act as an inhibitor of CPS-II, with an apparent Ki of 0.41 mM. However, 5-phospho-alpha-D-ribosyl-1-diphosphate, dimethyl sulphoxide and glycerol had no effect on the Km value for MgATP2-. The effect of some inhibitors, including pyrimidine and purine nucleotides and analogs and respiratory chain inhibitors, was also determined for the enzymes of the pathway.

Centrifugation, Density Gradient↗

PubMed2000

Engineering biosynthetic pathways: new routes to chiral amino acids.

The engineering of microbial biosynthetic pathways is advancing rapidly because of new molecular genetic approaches, sophisticated analysis of metabolic flux and the rapid sequencing of diverse bacterial genomes. The classical methods of mutagenesis/selection, originally applied in the development of amino-acid-over-producing bacteria are now being complemented by an increasingly rational strategy.

Amino Acids↗

PubMed2003

Sex pheromone biosynthetic pathway for disparlure in the gypsy moth, Lymantria dispar.

The pheromone biosynthetic pathway for production of the sex pheromone disparlure, 2-methyl-7R,8S-epoxy-octadecane, was determined for the gypsy moth. Each step in the pathway was followed by using deuterium-labeled compounds that could be identified by using GCMS. This approach provides unequivocal determination of specific reactions in the pathway. It was shown that the alkene precursor, 2-methyl-Z7-octadecene, is most likely made in oenocyte cells associated with abdominal epidermal cells. The pathway begins with valine contributing carbons for chain initiation, including the methyl-branched carbon, followed by chain elongation to 19 carbons. The double bond is introduced with an unusual Delta12 desaturase that utilizes a methyl-branched substrate. The resulting 18-methyl-Z12-nonadecenoate is decarboxylated to the hydrocarbon, 2-methyl-Z7-octadecene. The alkene is then transported to the pheromone gland through the hemolymph, most probably by lipophorin. At the pheromone gland, the alkene is unloaded and transformed into the epoxide disparlure for release into the environment. A chiral HPLC column was used to demonstrate that the (R,S)-stereoisomer of the epoxide, (+)-disparlure is found in pheromone glands.

Alkanes↗