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PubMed1996

Evidence for an Elongation/Reduction/C1-Elimination Pathway in the Biosynthesis of n-Heptane in Xylem of Jeffrey Pine.

The biosynthetic pathway to n-heptane was investigated by examining the effect of the [beta]-keto acyl-acyl carrier protein synthase inhibitor (2R,3S)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide (cerulenin), a thiol reagent ([beta]-mercaptoethanol), and an aldehydetrapping reagent (hydroxylamine) on the biosynthesis of n-[14C]heptane and putative intermediates in xylem sections of Jeffrey pine (Pinus jeffreyi Grev.& Balf.) incubated with [14C]acetate. Cerulenin inhibited C18 fatty acid biosynthesis but had relatively little effect on radiolabel incorporation into C8 fatty acyl groups and n-heptane. [beta]-Mercaptoethanol inhibited n-heptane biosynthesis, with a corresponding accumulation of radiolabel into both octanal and 1-octanol, whereas hydroxylamine inhibited both n-heptane and 1-octanol biosynthesis, with radiolabel accumulation in octyl oximes. [14C]Octanal was converted to both n-heptane and 1-octanol when incubated with xylem sections, whereas [14C]1-octanol was converted to octanal and n-heptane in a hydroxylamine-sensitive reaction. These results suggest a pathway for the biosynthesis of n-heptane whereby acetate is polymerized via a typical fatty acid synthase reaction sequence to yield a C8 thioester, which subsequently undergoes a two-electron reduction to generate a free thiol and octanal, the latter of which alternately undergoes an additional, reversible reduction to form 1-octanol or loss of C1 to generate n-heptane.

Journal Article↗

PubMed2000

Characterization of brassinazole, a triazole-type brassinosteroid biosynthesis inhibitor.

Screening for brassinosteroid (BR) biosynthesis inhibitors was performed to find chemicals that induce dwarfism in Arabidopsis, mutants that resembled BR biosynthesis mutants that can be rescued by BR. Through this screening experiment, the compound brassinazole was selected as the most potent chemical. In dark-grown Arabidopsis, brassinazole-induced morphological changes were nearly restored to those of wild type by treatment with brassinolide. The structure of brassinazole is similar to pacrobutrazol, a gibberellin biosynthesis inhibitor. However, in assays with cress (Lepidium sativum) plants, brassinazole-treated plants did not show recovery after the addition of gibberellin but showed good recovery after the addition of brassinolide. These data demonstrate that brassinazole is a specific BR biosynthesis inhibitor. Brassinazole-treated cress also showed dwarfism, with altered leaf morphology, including the downward curling and dark green color typical of Arabidopsis BR-deficient mutants, and this dwarfism was reversed by the application of 10 nM brassinolide. This result suggests that BRs are essential for plant growth, and that brassinazole can be used to clarify the function of BRs in plants as a complement to BR-deficient mutants. The brassinazole action site was also investigated by feeding BR biosynthesis intermediates to cress grown in the light.

Arabidopsis↗

PubMed1967

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips.

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-(14)C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a (14)C-product as IAA was by (a) co-chromatography of authentic IAA and (14)C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of alpha-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall congruent with etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.

Journal Article↗

PubMed1986

Paclobutrazol Inhibits Abscisic Acid Biosynthesis in Cercospora rosicola.

Three plant growth regulators, paclobutrazol, ancymidol, and decylimidazole, which are putative inhibitors of gibberellin (GA) biosynthesis, were studied to determine their effect on abscisic acid (ABA) biosynthesis in the fungus Cercospora rosicola. All three compounds inhibited ABA biosynthesis, and paclobutrazol was the most effective, inhibiting ABA 33% at 0.1 micromolar concentrations. In studies using (E,E,)-[1-(14)C] farnesyl pyrophosphate, it was shown that ancymidol blocked biosynthesis prior to farnesyl pyrophosphate (FPP), whereas paclobutrazol and decylimidazole acted after FPP. The three inhibitors did not prevent 4'-oxidation of (2Z,4E)-alpha-ionylideneacetic acid. C. rosiciola metabolized ancymidol by demethylation to alpha-cyclopropyl-alpha-(p-hydroxyphenyl)-5-pyrimidine methyl alcohol. Paclobutrazol was not metabolized by the fungus. Information that these plant growth regulators inhibit ABA as well as GA biosynthesis should prove useful in determining the full range of action of these compounds.

Journal Article↗

PubMed1980

Procollagen I synthesis in human skin fibroblasts: effect on culture conditions on biosynthesis.

Human skin fibroblast culture promises to be a useful system for the investigation of the regulation of collagen biosynthesis and the study of abnormalities in collagen biosynthesis in connective tissue disorders. The effect of culture conditions on procollagen I biosynthesis has been determined. Optimal conditions for collagen biosynthesis were: 10% dialyzed heat-inactivated fetal calf serum, 0.15 mM ascorbate, and 0.078 mM beta-aminopropionitrile. Newly synthesized procollagen I accumulated in the medium at a linear rate for 18 hr. Preincubation of cells in labelling media for 4 hr before adding radioactive proline enhanced synthesis. Collagenase digestion was used to study overall collagen biosynthesis. 94% of all collagen synthesized was found in the medium, and 6% in the cell pellet. Under optimal conditions, collagen comprised 24% of all protein in the medium, and 14% of protein produced by the whole culture.

Adolescent↗

PubMed1989

Differential effect of aspirin on thromboxane and prostaglandin biosynthesis in man.

1. Effects of a single intravenous dose of aspirin (600 mg) on bradykinin-stimulated prostaglandin (PG) and on thromboxane (TX) biosynthesis were determined in nine healthy male volunteers. Plasma concentrations of 6-oxo-PGF1 alpha and 13,14-dihydro-15-oxo-PGF2 alpha were measured in samples obtained during repeated 10 min intravenous infusions of bradykinin before and up to 6 h after the dose of aspirin. TXB2 was measured in serum from blood allowed to clot at 37 degrees C. 2. Aspirin inhibited bradykinin stimulated PG and platelet TX biosynthesis 0.5 h after the dose. Serum TXB2 remained low, whereas PG synthesis recovered within 6 h. 3. Effects of intravenous sodium salicylate (600 mg) were studied identically in eight subjects. Prostanoid biosynthesis was not inhibited. 4. Biosynthesis of prostacyclin and TXA2 under basal conditions was studied in eight subjects by measuring 2,3-dinor-6-oxo-PGF1 alpha and 2,3-dinor-TXB2 in hourly urine samples obtained during and after intravenous infusion of aspirin and, on a separate occasion, of vehicle. 5. Aspirin infusion reduced urinary excretion of both metabolites greater than 90%, but excretion of 2,3-dinor-6-oxo-PGF1 alpha recovered more rapidly than did that of 2,3-dinor-TXB2. 6. We conclude that cyclo-oxygenase is rapidly synthesised in bradykinin-responsive tissues in vivo and that this reflects similarly rapid enzyme biosynthesis in tissues that produce PGI2 under basal conditions.

6-Ketoprostaglandin F1 alpha↗

PubMed1990

Effects of non-steroidal anti-inflammatory drugs on prostacyclin and thromboxane biosynthesis in patients with mild essential hypertension.

1. The effects of non-steroidal anti-inflammatory drugs (NSAID) on prostacyclin and thromboxane biosynthesis and on blood pressure were determined in 46 patients with mild essential hypertension. Patients who had abstained from antihypertensive therapy for 2 weeks before study were treated with either aspirin, ibuprofen, sulindac or placebo for 7 days. 2. Excretion rates of 2,3-dinor-6-oxo-prostaglandin (PG) F1 alpha, 6-oxo-PGF1 alpha, 2,3-dinorthromboxane (TX) B2 and TXB2 were measured as indices of prostacyclin and TXA2 biosynthesis. Samples were assayed using immunoaffinity chromatography and gas chromatography/electron capture chemical ionisation mass spectrometry. 3. Aspirin and ibuprofen reduced urinary excretion of all prostacyclin- and thromboxane-derived products. Sulindac inhibited excretion of 2,3-dinor-6-oxo-PGF1 alpha, 6-oxo-PGF1 alpha and 2,3-dinor-TXB2, but had no significant effect on TXB2. 4. Systolic blood pressure increased in the ibuprofen-treated group when compared with the placebo group. There were no other significant changes in systolic or diastolic pressure in any of the treatment groups. Among the patients as a whole, there was a significant negative correlation between change in blood pressure and change in excretion of the prostacyclin-derived but not of the thromboxane-derived products. 5. We conclude that, in patients with mild essential hypertension, neither sulindac nor aspirin (in the doses used) selectively spares prostacyclin biosynthesis by the kidney. The significant relationship between increase in blood pressure and reduction in prostacyclin biosynthesis favours the possibility that in individuals who become hypertensive, prostacyclin biosynthesis determines, in part, the severity of the hypertensive state.

Adult↗

PubMed1995

A novel locus of Yersinia enterocolitica serotype O:3 involved in lipopolysaccharide outer core biosynthesis.

Yersinia enterocolitica serotype O:3 strain 6471/76-c (YeO3-c) was sensitive to bacteriophage phi R1-37 when grown at 37 degrees C but not when grown at 22 degrees C because of steric hindrance by abundant lipopolysaccharide (LPS) O-side chain (O-antigen) expressed at 22 degrees C. The transposon library of YeO3-c was grown at 37 degrees C and screened for phage phi R1-37-resistant transposon insertion mutants. Three types of mutant were isolated: (i) phage receptor mutants expressing O-antigen (LPS-smooth), (ii) phage receptor mutants not expressing O-antigen (LPS-rough), and (iii) LPS-smooth mutants with the phage receptor constitutively sterically blocked. Mutant type (i) was characterized in detail; the transposon insertion inactivates an operon, named the trs operon. The main findings based on this mutant are: (i) the frs operon is involved in the biosynthesis of the LPS outer core in YeO3-c; the nucleotide sequence of the trs operon revealed eight novel genes showing similarly to known polysaccharide biosynthetic genes of various Gram-negative bacteria as well as to capsule biosynthesis genes of Staphylococcus aureus; (ii) the biosynthesis of the core of YeO3-c involves at least two genetic loci; (iii) the trs operon is required for the biosynthesis of the bacteriophage phi R1-37 receptor structures; (iv) the homopolymeric O-antigen of YeO3-c is ligated to the inner core in Y. enterocolitica O:3; (v) the trs operon is located between the adk-hemH and galE-gsk gene pairs in the Y. enterocolitica chromosome; and (vi) the phage phi R1-37 receptor is present in many but not in all Y. enterocolitica serotypes. The results also allow us to speculate that the trs operon is a relic of the ancestral rfb region of Y. enterocolitica O:3 carrying genes indispensable for the completion of the core polysaccharide biosynthesis.

Amino Acid Sequence↗

PubMed2005

CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana.

Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the ROTUNDIFOLIA3 (ROT3) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in Arabidopsis thaliana, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to ROT3, CYP90D1, and found that double mutants for ROT3 and CYP90D1 had a severe dwarf phenotype, whereas cyp90d1 single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. ROT3 and CYP90D1 were expressed differentially in leaves of A. thaliana, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of CYP90D1 was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.

Amino Acid Sequence↗

PubMed1980

Glucosyltransferase activities in liver mitochondria. I. Biosynthesis of dolichyl[14C]glucosyl phosphate and [14C]glucosylceramide.

Mitochondria, and specially outer mitochondrial membranes, incorporate D-[14C]glucose from UDP-D-[14C]glucose into products extracted with organic solvents and into a residual precipitate, with a pH optimum of about 6.5 in (2-N-morpholino-ethane)-sulfonic acid (MES) buffer. The chloroform/methanol (2:1, v/v) extract contains two products. The major [14C]glucolipid is stable to mild alkali, but releases [14C]glucose upon mild acid hydrolysis. It is retained on DEAE-cellulose (acetate form) and is eluted with the same ionic strength as an hexosyldolichyl monophosphate diester. This [14C] glucolipid has the same chromatographic behaviour as dolichyl-mannosylphosphate in neutral, acidic and basic solvent systems; and its biosynthesis is greatly increased by exogenous dolichylmonophosphate. The other [14C]glucolipid is stable upon mild acid hydrolysis and is not retained on DEAE-cellulose. On silicic acid it is eluted with acetone. The biosynthesis of this compound is stimulated by exogenous ceramide. This glucolipid has the same chromatographic mobility in different solvent systems as glucosylceramide isolated from the liver of a patient with Gaucher's disease. Biosynthesis of these two glucolipids is inhibited by UDP, but only biosynthesis of dolichylglucosyl monophosphate is reversible with this nucleotide. The biosynthesis of these different glucosylated derivatives is stimulated by the addition of divalent cations (Mn2+, Mg2+). the effect of these two metal ions on dolichylglucosyl monophosphate and glucosylceramide formation is studied in different conditions.

Animals↗

PubMed1994

Biosynthesis of gangliosides containing C18:1 and C20:1 [3-14C]sphingosine after administrating [1-14C]palmitic acid and [1-14C]stearic acid to rat cerebellar granule cells in culture.

The biosynthesis of ganglioside molecular species containing sphingosine of different structure was investigated by administrating rat cerebellar granule cells in culture with [1-14C]palmitic and [1-14C]stearic acids which are the precursors for sphingosine biosynthesis. The incorporation of radioactivity into the sphingosine of the ganglioside species containing C20:1 sphingosine after administrating [1-14C]stearic acid was low in comparison with the incorporation of radioactivity into the sphingosine of ganglioside species containing C18:1 sphingosine after administration of [1-14C]palmitic acid, but the ratio between the radioactivity incorporated in the C20:1 and the C18:1 sphingosine of C20 and C18 ganglioside species progressively increased when the cell culture was prolonged. Ceramide-containing radioactive sphingosine was found after palmitic or stearic acid administration. Ceramide-containing C20:1 sphingosine found after adding stearic acid was about 5% of that synthesized starting from palmitic acid and containing C18:1 sphingosine. Free radioactive C18:1 and C20:1 sphingosine were found after adding radioactive palmitic or stearic acid. This is representative of a catabolic process occurring after biosynthesis of the complex sphingolipid starting from the radioactive precursor. In fact it has been proved that only saturated sphingosines are used for the synthesis of complex sphingolipids, the introduction of the double bond at position four of the sphingoid chain occurring at the level of ceramide [Rother, J., van Echten, G., Schwarzmann, G. & Sandhoff, K. (1992) Biochem. Biophys. Res. Commun. 189, 14-20]. Saturated sphingosines were not present. The lack of free C20:0 sphingosine confirms the hypothesis that the C20:0 sphingosine synthesis and the process (C20:0 sphingosine-->C20:0 ceramide-->C20:1 ceramide) occur in the correct quantity for the synthesis of C20:1 gangliosides. Moreover, we found only traces of free C20:1 sphingosine, at days 8 and 15 of cell culture when the biosynthesis of complex C20:1 gangliosides and the related catabolic processes occur to a higher extent, thus excluding the idea that a large amount of C20:0 sphingosine can be acylated to C20:0 ceramide and dehydrogenated to C20:1 ceramide which, being not used for ganglioside biosynthesis, is immediately catabolised to C20:1 sphingosine.

Animals↗

PubMed2006

Plant cell wall biosynthesis: genetic, biochemical and functional genomics approaches to the identification of key genes.

Cell walls are dynamic structures that represent key determinants of overall plant form, plant growth and development, and the responses of plants to environmental and pathogen-induced stresses. Walls play centrally important roles in the quality and processing of plant-based foods for both human and animal consumption, and in the production of fibres during pulp and paper manufacture. In the future, wall material that constitutes the major proportion of cereal straws and other crop residues will find increasing application as a source of renewable fuel and composite manufacture. Although the chemical structures of most wall constituents have been defined in detail, the enzymes involved in their synthesis and remodelling remain largely undefined, particularly those involved in polysaccharide biosynthesis. There have been real recent advances in our understanding of cellulose biosynthesis in plants, but, with few exceptions, the identities and modes of action of polysaccharide synthases and other glycosyltransferases that mediate the biosynthesis of the major non-cellulosic wall polysaccharides are not known. Nevertheless, emerging functional genomics and molecular genetics technologies are now allowing us to re-examine the central questions related to wall biosynthesis. The availability of the rice, Populus trichocarpa and Arabidopsis genome sequences, a variety of mutant populations, high-density genetic maps for cereals and other industrially important plants, high-throughput genome and transcript analysis systems, extensive publicly available genomics resources and an increasing armoury of analysis systems for the definition of candidate gene function will together allow us to take a systems approach to the description of wall biosynthesis in plants.

Biotechnology↗

PubMed1985

Cholesterol biosynthesis and its regulation in dissociated cell cultures of fetal rat brain: developmental changes and the role of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.

Animals↗

PubMed2006

Cannabinoids attenuate norepinephrine-induced melatonin biosynthesis in the rat pineal gland by reducing arylalkylamine N-acetyltransferase activity without involvement of cannabinoid receptors.

Cannabinoids modulate neuronal and neuroendocrine circuits by binding to cannabinoid receptors acting upon cAMP/Ca(2+)-mediated intracellular signaling cascades. The rat pineal represents an established model to investigate intracellular signaling processes because a well defined input, the neurotransmitter norepinephrine, is transformed via cAMP/Ca(2+)-dependent mechanisms into an easily detectable output signal, the biosynthesis of melatonin. Here we investigated the impact of cannabinoids on norepinephrine-regulated melatonin biosynthesis in the rat pineal. We demonstrated that treatment of cultured rat pineals with 9-carboxy-11-nor-delta-9-tetrahydrocannabinol (THC), cannabidiol or cannabinol significantly reduced norepinephrine-induced arylalkylamine N-acetyltransferase (AANAT) activity and melatonin biosynthesis. These effects were not mimicked by the cannabinoid receptor agonist WIN55,212-2 and were not blocked by cannabinoid 1 and 2 receptor antagonists. The cannabinoids used did not affect norepinephrine-induced increases in cAMP/Ca(2+) levels. Notably, cannabinoids were found to directly inhibit AANAT activity in lysates of the pineal gland. This effect was specific in so far as cannabinoids did not influence the activity of hydroxyindole-O-methyltransferase (HIOMT), the last enzyme in melatonin biosynthesis. Taken together, our data strongly suggest that cannabinoids inhibit AANAT activity and attenuate melatonin biosynthesis through intracellular actions without involvement of classical cannabinoid receptor-dependent signaling cascades.

Animals↗

PubMed1987

Isoprenoid biosynthesis in multiple sclerosis, II. A possible role of NADPH.

Genetic predisposition in MS, influence of fat consumption on the disease, and excretion of lipid metabolites in urine led us to investigate isoprenoid metabolism in this disease. Ubiquinone concentration and biosynthesis was normal in lymphocytes. Cytochrome oxidase, which contains an isoprenoid side chain, was normal in activity. Cholesterol biosynthesis from acetate was found to be elevated in MS, and so was triglyceride biosynthesis. Increased biosynthesis may offer a very simple explanation to all the metabolites excreted (3-methylglutaconic acid, 2-hydroxy-2-methyl-3-butenoic acid and adipic acid). Increased biosynthesis may be caused by an elevated NADPH/NADP ratio, since such an elevation may also account for many other biochemical anomalies in MS. Elevated NADPH/NADP ratio may be of direct importance in the pathogenesis.

Cholesterol↗

PubMed1999

CREB phosphorylation and melatonin biosynthesis in the rat pineal gland: involvement of cyclic AMP dependent protein kinase type II.

Phosphorylation of cyclic AMP response element binding protein (CREB) at amino acid serine 133 appears as an important link between the norepinephrine (NE)-induced activation of second messenger systems and the stimulation of melatonin biosynthesis. Here we investigated in the rat pineal gland: 1) the type of protein kinase that mediates CREB phosphorylation: and 2) its impact on melatonin biosynthesis. Immunochemical or immunocytochemical demonstration of serine133-phosphorylated cyclic AMP regulated element binding protein (pCREB) and radioimmunological detection of melatonin revealed that only cyclic AMP-dependent protein kinase (PKA) inhibitors suppressed NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis, whereas inhibitors of cyclic GMP-dependent protein kinase (PKG), mitogen-activated protein kinase kinase, protein kinase C, or calcium-calmodulin-dependent protein kinase (CaMK) were ineffective. Investigations with cyclic AMP-agonist pairs that selectively activate either PKA type I or II link NE-induced CREB phosphorylation and stimulation of melatonin biosynthesis to the activation of PKA type II. Our data suggest that PKA type II plays an important role in the transcriptional control of melatonin biosynthesis in the rat pineal organ.

Animals↗

PubMed2006

Structure and biosynthesis of heat-stable antifungal factor (HSAF), a broad-spectrum antimycotic with a novel mode of action.

A screen for antifungal compounds from Lysobacter enzymogenes strain C3, a bacterial biological control agent of fungal diseases, has previously led to the isolation of heat-stable antifungal factor (HSAF). HSAF exhibits inhibitory activities against a wide range of fungal species and shows a novel mode of antifungal action by disrupting the biosynthesis of a distinct group of sphingolipids. We have now determined the chemical structure of HSAF, which is identical to that of dihydromaltophilin, an antifungal metabolite with a unique macrocyclic lactam system containing a tetramic acid moiety and a 5,5,6-tricyclic skeleton. We have also identified the genetic locus responsible for the biosynthesis of HSAF in strain C3. DNA sequencing of this locus revealed genes for a hybrid polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS), a sterol desaturase, a ferredoxin reductase, and an arginase. The disruption of the PKS-NRPS gene generated C3 mutants that lost the ability to produce HSAF and to inhibit fungal growth, demonstrating a hybrid PKS-NRPS that catalyzed the biosynthesis of the unique macrolactam system that is found in many biologically active natural products isolated from marine organisms. In addition, we have generated mutants with disrupted sterol desaturase, ferredoxin reductase, and arginase and examined the metabolites produced in these mutants. The work represents the first study of the genetic basis for the biosynthesis of the tetramic acid-containing macrolactams. The elucidation of the chemical structure of HSAF and the identification of the genetic locus for its biosynthesis establish the foundation for future exploitation of this group of compounds as new fungicides or antifungal drugs.

Amides↗

PubMed1980

Metabolic regulation in tylosin-producing Streptomyces fradiae: regulatory role of adenylate nucleotide pool and enzymes involved in biosynthesis of tylonolide precursors.

The relationship was studied between the level of the intracellular adenylates and the biosynthesis of tylosin by Streptomyces fradiae NRRL 2702. The adenylate level was observed to be inversely related to the rate of tylosin biosynthesis and hence the final concentration of the antibiotic. The concentrations of the adenylates were maximal during the trophophase, dropped quickly before the onset of tylosin biosynthesis, and remained at low levels throughout the idiophase. The adenylate energy charge was almost constant throughout the fermentation and was in the range of 0.4 to 0.55. Glucose addition in the idiophase suppressed tylosin biosynthesis, accompanied by a rapid increase in the adenylate levels. The biosynthesis of tylosin resumed after a rapid drop in the adenosine triphosphate concentration. Two enzymes catalyzing the interconversion of propionyl-coenzyme A and methylmalonyl-coenzyme A were found in this organism: methylmalonyl-coenzyme A carboxyltransferase (EC 2.1.3.1) and propionyl-coenzyme A carboxylase (EC 6.4.1.3). The activity of the former was two orders of magnitude higher than that of the latter. The activities of both enzymes were affected by the increased glucose addition in the idiophase.

Adenine Nucleotides↗