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PubMed2006

Mechanistic possibilities in MauG-dependent tryptophan tryptophylquinone biosynthesis.

Tryptophan tryptophylquinone (TTQ), the prosthetic group of methylamine dehydrogenase, is formed by post-translational modifications of two tryptophan residues that result in the incorporation of two oxygens into one tryptophan side chain and the covalent cross-linking of that side chain to a second tryptophan residue. MauG is a novel 42 kDa di-heme protein, which is required for the biosynthesis of TTQ. An experimental system has been developed that allows the direct continuous monitoring of MauG-dependent TTQ biosynthesis in vitro. Four diverse electron donors, ascorbate, dithiothreitol, reduced glutathione, and NADH, were each able to provide reducing equivalents for MauG-dependent TTQ biosynthesis under aerobic conditions. The reaction with NADH was mediated by an NADH-dependent oxidoreductase. Under anaerobic conditions in the absence of an electron donor, H(2)O(2) could serve as a substrate for MauG-dependent TTQ biosynthesis. During the reaction with H(2)O(2), a discrete reaction intermediate was observed, which is likely the reduced quinol form of TTQ that then is oxidized to the quinone. These results suggest that not only the incorporation of oxygen into the monohydroxylated biosynthetic intermediate but also the subsequent oxidation of quinol MADH during TTQ biosynthesis is a MauG-dependent process. The implications of these results in elucidating the mechanism of MauG-dependent TTQ biosynthesis and identifying potential physiologic electron and oxygen donors for TTQ biosynthesis in vivo are discussed.

Electrons↗
PubMed2004

Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP.

1. Histamine is generally regarded as a pro-inflammatory mediator in diseases such as allergy and asthma. A growing number of studies, however, suggest that this autacoid is also involved in the downregulation of human polymorphonuclear leukocyte (PMN) functions and inflammatory responses through activation of the Gs-coupled histamine H(2) receptor. 2. We report here that histamine inhibits thapsigargin- and ligand (PAF and fMLP)-induced leukotriene (LT) biosynthesis in human PMN in a dose-dependent manner. 3. The suppressive effect of histamine on LT biosynthesis was abrogated by the histamine H(2) receptor antagonists cimetidine, ranitidine, and tiotidine. In contrast, the histamine H(1), H(3), and H(4) receptor antagonists used in this study were ineffective in counteracting the inhibitory effect of histamine on the biosynthesis of LT in activated human PMN. 4. The inhibition of LT biosynthesis by histamine was characterized by decreased arachidonic acid release and 5-lipoxygenase translocation to the nuclear membrane. 5. Incubation of PMN with the cAMP-dependent protein kinase (PKA) inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide prevented the inhibitory effect of histamine on LT biosynthesis, suggesting an important role for PKA in this effect of histamine on LT biosynthesis in PMN. 6. These data provide the first evidences that, similarly to adenosine and prostaglandin E(2), histamine is a potent suppressor of LT biosynthesis, and support the concept that histamine may play a dual role in the regulation of inflammation.

Arachidonate 5-Lipoxygenase↗
PubMed2006

Effects of pyrrophenone, an inhibitor of group IVA phospholipase A2, on eicosanoid and PAF biosynthesis in human neutrophils.

BACKGROUND AND PURPOSE: The biosynthesis of leukotrienes (LT) and platelet-activating factor (PAF) involves the release of their respective precursors, arachidonic acid (AA) and lyso-PAF by the group IVA PLA2 (cPLA2alpha). This paper aims at characterizing the inhibitory properties of the cPLA2alpha inhibitor pyrrophenone on eicosanoids and PAF in human neutrophils (PMN). EXPERIMENTAL APPROACH: Freshly isolated human PMN were activated with physiological and pharmacological agents (fMLP, PAF, exogenous AA, A23187 and thapsigargin) in presence and absence of the cPLA2alpha inhibitor pyrrophenone and biosynthesis of LT, PAF, and PGE2 was measured. KEY RESULTS: Pyrrophenone potently inhibited LT, PGE2 and PAF biosynthesis in PMN with IC50s in the range of 1-20 nM. These inhibitory effects of pyrrophenone were specific (the consequence of substrate deprivation), as shown by the reversal of inhibition by exogenous AA and lyso-PAF. Comparative assessment of pyrrophenone, methyl-arachidonoyl-fluoro-phosphonate (MAFP) and arachidonoyl-trifluoromethylketone (AACOCF3) demonstrated that pyrrophenone was more specific and 100-fold more potent than MAFP and AACOCF3 for the inhibition of LT biosynthesis in A23187-activated PMN. The inhibitory effect of pyrrophenone on LT biosynthesis was reversible as LT biosynthesis was recovered when pyrrophenone-treated PMN were washed with autologous plasma. No alteration of phospholipase D (PLD) activity in fMLP-activated PMN was observed with up to 10 microM pyrrophenone, suggesting that the cPLA2alpha inhibitor does not directly inhibit PLD. CONCLUSIONS AND IMPLICATIONS: Pyrrophenone is a more potent and specific cPLA2alpha inhibitor than MAFP and AACOCF3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of eicosanoids and PAF.

Arachidonic Acids↗
PubMed1978

Control of amylase biosynthesis and release in the parotid gland of the rat.

1. Amylase biosynthesis and release in the rat parotid were studied under various conditions. Incorporation of [(3)H]leucine into amylase, extracted from the tissue by immunoadsorbent, was measured and found to be time-dependent and totally inhibited by the protein synthesis inhibitor puromycin. 2. Adrenaline, at a concentration (10mum) that gave maximum stimulation of release, inhibited [(3)H]leucine incorporation into both total protein and amylase. This effect was reversed by phentolamine. 3. Adrenaline (1mum) and isoproterenol (10mum) stimulated biosynthesis of total protein and amylase. These effects were blocked by propranolol, as were the effects on release. Dibutyryl cyclic AMP (2mm) mimicked the effects of isoproterenol and adrenaline (1mum) on both amylase biosynthesis and release. All the above stimulatory effects on amylase biosynthesis were only observed if the tissue was pretreated with effector before pulse-labelling with [(3)H]leucine. 4. Insulin (625muunits/ml initial concentration, 150muunits/ml final concentration) stimulated incorporation of [(3)H]leucine into total protein and amylase when added to the tissue at the same time as the leucine. 5. Carbamoylcholine (10mum) decreased [(3)H]leucine incorporation into total protein and amylase when both were added to the tissue simultaneously, but this effect was prevented by removal of effector and washing the tissue before addition of [(3)H]leucine. 6. Stimulation of beta-adrenergic receptors increased both amylase release and biosynthesis, but stimulation of alpha-receptors can inhibit biosynthesis without inhibiting release. Cholinergic agents can also inhibit amylase biosynthesis, but stimulate release. Insulin at approximately physiological concentration can increase incorporation of leucine into amylase without stimulating release. The system described therefore provides an excellent model for the further investigation of the mechanisms of these diverse effects.

Amylases↗
PubMed1983

Glutathione biosynthesis in the aging adult yellow-fever mosquito [Aedes aegypti (Louisville)].

Our previous findings [Hazelton & Lang (1978) Fed. Proc. Fed. Am. Soc. Exp. Biol. 37(6), 2378 (abstr.)] demonstrated a senescence-specific decrease in glutathione (GSH) concentration in the yellow-fever mosquito Aedes aegypti (Louisville)]. As a possible mechanism for this change, GSH biosynthesis was investigated in adult mosquitoes of different ages through the life-span. Biosynthesis was measured as the incorporation rate of [14C]glycine or [14C]cystine into glutathione. Essential information to validate the procedure was also obtained on the precursor-amino-acid pool sizes and kinetic parameters such as lag-time and time course of incorporation. Also, synthesis de novo rather than exchange was verified using buthionine sulphoximine, a specific inhibitor of GSH biosynthesis. The synthetic rates with either amino acid precursor varied throughout the adult life-span, but the patterns for both precursors were essentially identical. Biosynthesis was high in the newly emerged adult and decreased 62-70% (P less than 0.005) to a plateau during maturity. From the mature value there was a decrease of 36-41% (P less than 0.005) to a new plateau during senescence. Glutathione biosynthesis and concentration were correlated throughout maturity and senescence (r = 0.982) and thus biosynthesis was proportional to glutathione content. On this basis we concluded that impaired biosynthesis is the major and perhaps sole mechanism for the aging decrease in glutathione content.

Aedes↗
PubMed1986

Erythrocyte-neutrophil interactions: formation of leukotriene B4 by transcellular biosynthesis.

Studies on the mechanism of leukotriene B4 biosynthesis in suspensions composed of neutrophils plus erythrocytes indicate that human erythrocytes convert neutrophil-derived leukotriene A4 into leukotriene B4. Leukotriene B4 formation by neutrophils in the presence of erythrocytes exceeded that from corresponding suspensions of neutrophils alone. The increase was proportional to the erythrocyte content of the suspension. The erythrocyte-dependent increase in leukotriene B4 biosynthesis did not equal the arithmetic sum of calcium ionophore-dependent biosynthesis by neutrophils plus calcium ionophore-dependent biosynthesis by erythrocytes, since erythrocytes produced no leukotriene B4 upon incubation with ionophore A23187. Erythrocytes did not stimulate 5-lipoxygenase activity within neutrophils, since the erythrocyte effect was confined to enzymatic hydration: leukotriene B4 increased coincident with decreased formation of 5,12-dihydroxyicosatetraenoic acids derived from nonenzymatic hydration. Biosynthesis of leukotriene B4 within the erythrocyte, from neutrophil-derived leukotriene A4, was established by comparing the effect of normal erythrocytes with erythrocytes containing a leukotriene A4 hydrolase that was inactivated by the substrate. In the latter case, leukotriene B4 formation increased by only 30-40%; in the former case, it increased by 100-200%. Transcellular biosynthesis of leukotriene B4 from erythrocyte-neutrophil interactions explains the paradoxical presence of leukotriene A4 hydrolase within erythrocytes, a cell incapable of synthesizing leukotriene A4; affords a mechanism to overcome rate limitations or "suicide inactivation" of leukotriene A4 hydrolase in neutrophils; exploits a cryptic capacity within erythrocytes, provisionally dormant cells in terms of icosanoid biosynthesis; indicates that the biosynthetic capacity of cell combinations is not necessarily equivalent to the sum of their separate capacities.

Arachidonic Acids↗
PubMed1997

cis-4-Methylsphingosine decreases sphingolipid biosynthesis by specifically interfering with serine palmitoyltransferase activity in primary cultured neurons.

The effect of six different structurally modified sphingosine analogues on biosynthesis of sphingolipids was studied in primary cultured murine cerebellar neurons. Treatment of cells with cis-4-methylsphingosine at micromolar levels resulted in a markedly decreased sphingolipid biosynthesis, whereas the other compounds examined, trans-4-methylsphingosine, cis-5-methylsphingosine, trans-5-methylsphingosine, cis-sphingosine, and 1-deoxysphingosine, inhibited sphingolipid biosynthesis less efficiently. The inhibition of sphingolipid biosynthesis by the various compounds was paralleled by a decrease of serine palmitoyltransferase activity in situ. For cis-4-methylsphingosine the inhibitory effect on serine palmitoyltransferase activity was shown to be concentration- and time-dependent. Half-maximal reduction of enzyme activity occurred after 24 h of treatment with 10 microM of the compound. The activity of other enzymes of sphingolipid biosynthesis as well as phospholipid and protein biosynthesis was not affected. Analysis of the sphingoid moiety of cellular sphingolipids suggests that the sphingosine analogues listed above were subject to degradation rather than being utilized as precursors for sphingolipid biosynthesis by cultured neurons. Except of 1-deoxysphingosine, the other five sphingosine analogues were shown to be substrates for sphingosine kinase in vitro. After 24 h of treatment of primary cerebellar neurons with the various sphingosine analogues the relative percentage of the respective intracellular 1-phosphate derivatives paralleled exactly the inhibitory effect on serine palmitoyltransferase activity observed when cells were treated with the unphosphorylated compounds. In contrast to the respective 1-phosphate derivatives of the other methyl-branched sphingosine analogues examined, cis-4-methylsphingosine 1-phosphate showed an intracellular accumulation suggesting a delayed turnover rate in cultured murine neurons for this compound. These results suggest that the inhibitory effect of the sphingosine analogues on serine palmitoyltransferase is mediated by their respective 1-phosphate derivatives and that the pronounced effect of cis-4-methylsphingosine is caused by a high intracellular concentration of cis-4-methylsphingosine 1-phosphate. cis-4-Methylsphingosine, in addition, caused drastic changes in cell morphology of primary cerebellar neurons, which were not observed when these cells were treated with one of the other sphingosine analogues examined.

Acyltransferases↗
PubMed1999

Bacterioopsin-triggered retinal biosynthesis is inhibited by bacteriorhodopsin formation in Halobacterium salinarium.

Factors regulating retinal biosynthesis in halobacteria are not clearly understood. In halobacteria, events leading to the biosynthesis of bacteriorhodopsin have been proposed to participate in stringent regulation of retinal biosynthesis. The present study describes a novel approach of in vivo introductions of mRNA and membrane proteins via liposome fusion to test their role in cellular metabolism. Both the bacterioopsin-encoding mRNA and the liposome-encapsulated bacterioopsin (apoprotein) are independently introduced in spheroplasts of the purple membrane-negative strain Halobacterium salinarium that initially contain neither bacterioopsin nor retinal. Isoprenoid analyses of these cells indicate that the expression/presence of bacterioopsin triggers retinal biosynthesis from lycopene, and its subsequent binding to opsin generates bacteriorhodopsin. When bacteriorhodopsin and excess retinal were independently introduced into spheroplasts of purple membrane-negative cells, the introduction of bacteriorhodopsin resulted in an accumulation of lycopene, indicating an inhibition of retinal biosynthesis. These results provide direct evidence that the formation of bacterioopsin acts as a trigger for lycopene conversion to beta-carotene in retinal biosynthesis. The trigger for this event does not lie with either transcription or translation of the bop gene. It is clearly associated with the folded and the membrane-integrated state of bacterioopsin. On the other hand, the trigger signaling inhibition of retinal biosynthesis does not lie with the presence of excess retinal but with the correctly folded, retinal-bound form, bacteriorhodopsin.

Amino Acid Sequence↗
PubMed2001

Saccharomyces cerevisiae is capable of de Novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine.

Pantothenic acid and beta-alanine are metabolic intermediates in coenzyme A biosynthesis. Using a functional screen in the yeast Saccharomyces cerevisiae, a putative amine oxidase, encoded by FMS1, was found to be rate-limiting for beta-alanine and pantothenic acid biosynthesis. Overexpression of FMS1 caused excess pantothenic acid to be excreted into the medium, whereas deletion mutants required beta-alanine or pantothenic acid for growth. Furthermore, yeast genes ECM31 and YIL145c, which both have structural homology to genes of the bacterial pantothenic acid pathway, were also required for pantothenic acid biosynthesis. The homology of FMS1 to FAD-containing amine oxidases and its role in beta-alanine biosynthesis suggested that its substrates are polyamines. Indeed, we found that all the enzymes of the polyamine pathway in yeast are necessary for beta-alanine biosynthesis; spe1Delta, spe2Delta, spe3Delta, and spe4Delta are all beta-alanine auxotrophs. Thus, contrary to previous reports, yeast is naturally capable of pantothenic acid biosynthesis, and the beta-alanine is derived from methionine via a pathway involving spermine. These findings should facilitate the identification of further enzymes and biochemical pathways involved in polyamine degradation and pantothenic acid biosynthesis in S. cerevisiae and raise questions about these pathways in other organisms.

Pantothenic Acid↗
PubMed2001

Basic fibroblast growth factor-induced proliferation of primary astrocytes. evidence for the involvement of sphingomyelin biosynthesis.

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either l-[(3)H]serine or [(3)H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.

Animals↗
PubMed1999

Lanosterol analogs: dual-action inhibitors of cholesterol biosynthesis.

Drugs which suppress hepatic cholesterol biosynthesis are important therapeutic tools for lowering serum cholesterol, a major risk factor in coronary heart disease. With the goal of developing molecules that will effectively shut down cholesterol biosynthesis in hepatic tissue but allow for the buildup of the isoprenes needed for the biosynthesis of polyisoprenes other than sterols, we have designed and evaluated a series of lanosterol analogs to act as dual-action inhibitors of cholesterol biosynthesis. These sterols were predicted to act as competitive inhibitors of lanosterol 14alpha-methyl demethylase (P-450DM) and as partial suppressors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), the rate-limiting enzyme in the pathway. Compounds which have been identified as dual-action inhibitors of cholesterol biosynthesis include analogs of the intermediates generated during the removal of the 14alpha-methyl group of lanosterol by P-450DM, aminolanosterols with the amine nitrogen placed in the vicinity of C-32, and lanosterol analogs with a ketone or oxime functionality at C-15. While some dual-action inhibitors require an active P-450DM for suppression of HMGR activity, others do not. The inability of some compounds to suppress HMGR activity in cells which lack P-450DM activity suggests either that these compounds require P-450DM for conversion to an active metabolite which then suppresses HMGR activity, or that they cause the accumulation of the natural demethylation intermediates resulting in the suppression of HMGR activity. Lanosterol analogs, in contrast to 25-hydroxycholesterol, do not inhibit transcription of the HMGR gene. Rather, they inhibit translation of the HMGR mRNA, and in most cases also accelerate the degradation of enzyme protein. The potential pharmacological utility of cholesterol biosynthesis inhibitors may be determined at least in part by their effects on LDL receptor (LDLR) activity. The transcriptional regulator 25-hydroxycholesterol suppresses both HMGR and LDLR activities, while the post-transcriptional regulatory lanosterol analogs exhibit a more desirable profile, lowering HMGR levels without suppressing LDLR expression, and in some cases actually enhancing cellular LDL metabolism. Lanosterol analogs which function as dual-action inhibitors of cholesterol biosynthesis promise to be useful not only as tools for dissecting the cellular regulation of cholesterol metabolism, but also as models for the development of safe, effective hypocholesterolemic agents.

Animals↗
PubMed1989

POMC biosynthesis in the intermediate lobe of the spontaneously hypertensive rat.

Pro-opiomelanocortin (POMC) synthesized in the anterior (AL) and intermediate lobe (IL) of the rat pituitary gland, is a prohormone precursor of several peptide hormones that may participate in the regulation of blood pressure. We therefore studied the biosynthesis of POMC by measuring the incorporation of 3H-tyrosine into the IL of 32-week-old spontaneously hypertensive rat (SHR) of the Aoki-Okamoto strain and their normotensive controls, the Wistar-Kyoto rat (WKY). Under basal conditions, POMC biosynthesis was significantly reduced in the SHR (1130 +/- 102 cpm/30 min/IL, n = 13) when compared to the WKY (1515 +/- 163 cpm/30 min/IL, N = 12, P less than 0.05, t test). There was also an inverse correlation between systolic blood pressure and POMC biosynthesis in both the WKY (Y = 8.4 +/- 1.38X + 2097 +/- 673, r = 0.86 +/- 0.06, N = 5), and SHR (Y = 5.7 +/- 1.8X + 1122 +/- 336 r = 0.57 +/- 0.13, N = 3). The decreased POMC biosynthesis was associated with a higher dopamine content in IL in SHR compared to WKY (1.56 +/- 0.53, n = 17 v 0.51 +/- 0.16, n = 17 pmol/IL, P less than 0.05, Mann-Whitney test). Oral administration of three different antihypertensive agents (propranolol, captopril, or hydralazine) for 12 weeks normalized blood pressure and POMC biosynthesis in the SHR but had no effect on either variable in the WKY. Because POMC biosynthesis in IL increased with normalization of blood pressure in the SHR, the decreased POMC biosynthesis in SHR may be a consequence rather than a cause of the elevated blood pressure in SHR.

Animals↗
PubMed2003

Efficiency of lignin biosynthesis: a quantitative analysis.

Lignin is derived mainly from three alcohol monomers: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Biochemical reactions probably responsible for synthesizing these three monomers from sucrose, and then polymerizing the monomers into lignin, were analysed to estimate the amount of sucrose required to produce a unit of lignin. Included in the calculations were amounts of respiration required to provide NADPH (from NADP(+)) and ATP (from ADP) for lignin biosynthesis. Two pathways in the middle stage of monomer biosynthesis were considered: one via tyrosine (found in monocots) and the other via phenylalanine (found in all plants). If lignin biosynthesis proceeds with high efficiency via tyrosine, 76.9, 70.4 and 64.3 % of the carbon in sucrose can be retained in the fraction of lignin derived from p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, respectively. The corresponding carbon retention values for lignin biosynthesis via phenylalanine are less, at 73.2, 65.7 and 60.7 %, respectively. Energy (i.e. heat of combustion) retention during lignin biosynthesis via tyrosine could be as high as 81.6, 74.5 and 67.8 % for lignin derived from p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol, respectively, with the corresponding potential energy retention values for lignin biosynthesis via phenylalanine being less, at 77.7, 69.5 and 63.9 %, respectively. Whether maximum efficiency occurs in situ is unclear, but these values are targets that can be considered in: (1) plant breeding programmes aimed at maximizing carbon or energy retention from photosynthate; (2) analyses of (minimum) metabolic costs of responding to environmental change or pest attack involving increased lignin biosynthesis; (3) understanding costs of lignification in older tissues; and (4) interpreting carbon balance measurements of organs and plants with large lignin concentrations.

Biological Transport↗
PubMed2025

ODR1, the key seed dormancy and germination regulator, promotes seed Proanthocyanidin biosynthesis via interaction with TTG1 and modulation of MBW complex activity.

Seed dormancy and germination are crucial for both plant survival and reproduction and for crop sowing and harvesting. Proanthocyanidins (PAs), one of the most abundant seed metabolites, play a role in enhancing dormancy and inhibiting germination. Multiple regulatory factors involved in PAs biosynthesis can alter seed dormancy or germination capacity. However, whether the dormancy or germination factors reciprocally influence the PAs biosynthesis is unclear. Here, we report that ODR1, a seed dormancy and germination key factor and a transcriptional (co-) repressor, can regulate seed PAs biosynthesis and act as a transcriptional co-activator. The odr1 mutant shows lighter seed coat color, decreased PAs contents, and reduced expression of PAs biosynthesis genes, which are restored in the ODR1 complementary lines. ODR1 interacts with TTG1 and forms a complex with TTG1/TT2/TT8 (three MBW complex components), enhancing their activation on promoters of PAs biosynthesis genes like DFR and ANS. Overexpressing TTG1 in the odr1-2 mutant rescues or even reverses PA-related phenotypes of odr1-2, confirming that ODR1-mediated regulation of PAs biosynthesis is dependent on TTG1. Moreover, three homologous copies of ODR1 in rapeseed were identified, and simultaneous knockout of them reduces the PAs contents. These results revealed the previously uncharacterized functions of ODR1 in PAs biosynthesis, suggested its conservation between Arabidopsis and rapeseed, and provided important gene resources for rapeseed variety improvement.

Proanthocyanidins↗
PubMed2006

Isolation, characterization, and heterologous expression of the biosynthesis gene cluster for the antitumor anthracycline steffimycin.

The biosynthetic gene cluster for the aromatic polyketide steffimycin of the anthracycline family has been cloned and characterized from "Streptomyces steffisburgensis" NRRL 3193. Sequence analysis of a 42.8-kbp DNA region revealed the presence of 36 open reading frames (ORFs) (one of them incomplete), 24 of which, spanning 26.5 kb, are probably involved in steffimycin biosynthesis. They code for all the activities required for polyketide biosynthesis, tailoring, regulation, and resistance but show no evidence of genes involved in L-rhamnose biosynthesis. The involvement of the cluster in steffimycin biosynthesis was confirmed by expression of a region of about 15 kb containing 15 ORFS, 11 of them forming part of the cluster, in the heterologous host Streptomyces albus, allowing the isolation of a biosynthetic intermediate. In addition, the expression in S. albus of the entire cluster, contained in a region of 34.8 kb, combined with the expression of plasmid pRHAM, directing the biosynthesis of L-rhamnose, led to the production of steffimycin. Inactivation of the stfX gene, coding for a putative cyclase, revealed that this enzymatic activity participates in the cyclization of the fourth ring, making the final steps in the biosynthesis of the steffimycin aglycon similar to those in the biosynthesis of jadomycin or rabelomycin. Inactivation of the stfG gene, coding for a putative glycosyltransferase involved in the attachment of L-rhamnose, allowed the production of a new compound corresponding to the steffimycin aglycon compound also observed in S. albus upon expression of the entire cluster.

Anthracyclines↗
PubMed1976

Macromolecular syntheses during biosynthesis of prodigiosin by Serratia marcescens.

Amino acids that were utilized as sole sources of carbon and nitrogen for growth of Serratia marcescens Nima resulted in biosynthesis of prodigiosin in non-proliferating bacteria. Addition of alanine, proline, or histidine to non-proliferating cells incubated at 27 C increased the rate of protein synthesis and also caused biosynthesis of prodigiosin. No increase in the rate of protein synthesis was observed upon the addition of amino acids that did not stimulate prodigiosin biosynthesis. Increased rates of synthesis of ribonucleic acid (RNA) and of deoxyribonucleic acid (DNA) (a small amount) also occurred after addition of amino acids that resulted in biosynthesis of prodigiosin. After incubation of 24 h, the total amount of protein in suspensions of bacteria to which alanine or proline was added increased 67 and 98%, respectively. Total amounts of DNA and of RNA also increased before synthesis of prodigiosin. The amounts of these macromolecules did not increase after addition of amino acids that did not induce biosynthesis of progidiosin. However, macromolecular synthesis was not related only to prodigiosin biosynthesis because the rates of DNA, RNA, and protein synthesis also increased in suspensions of bacteria incubated with proline at 39 C, at which temperature no prodigiosin was synthesized. The quantities of DNA, RNA, and protein synthesized were lower in non-proliferating cells than in growing cells. The data indicated that amino acids causing biosynthesis of prodigiosin in non-proliferating cells must be metabolized and serve as sources of carbon and of nitrogen for synthesis of macromolecules and intermediates. Prodigiosin was synthesized secondarily to these primary metabolic events.

Amino Acids↗
PubMed1997

Domains of Escherichia coli acyl carrier protein important for membrane-derived-oligosaccharide biosynthesis.

Acyl carrier protein participates in a number of biosynthetic pathways in Escherichia coli: fatty acid biosynthesis, phospholipid biosynthesis, lipopolysaccharide biosynthesis, activation of prohemolysin, and membrane-derived oligosaccharide biosynthesis. The first four pathways require the protein's prosthetic group, phosphopantetheine, to assemble an acyl chain or to transfer an acyl group from the thioester linkage to a specific substrate. By contrast, the phosphopantetheine prosthetic group is not required for membrane-derived oligosaccharide biosynthesis, and the function of acyl carrier protein in this biosynthetic scheme is currently unknown. We have combined biochemical and molecular biological approaches to investigate domains of acyl carrier protein that are important for membrane-derived oligosaccharide biosynthesis. Proteolytic removal of the first 6 amino acids from acyl carrier protein or chemical synthesis of a partial peptide encompassing residues 26 to 50 resulted in losses of secondary and tertiary structure and consequent loss of activity in the membrane glucosyltransferase reaction of membrane-derived oligosaccharide biosynthesis. These peptide fragments, however, inhibited the action of intact acyl carrier protein in the enzymatic reaction. This suggests a role for the loop regions of the E. coli acyl carrier protein and the need for at least two regions of the protein for participation in the glucosyltransferase reaction. We have purified acyl carrier protein from eight species of Proteobacteria (including representatives from all four subgroups) and characterized the proteins as active or inhibitory in the membrane glucosyltransferase reaction. The complete or partial amino acid sequences of these acyl carrier proteins were determined. The results of site-directed mutagenesis to change amino acids conserved in active, and altered in inactive, acyl carrier proteins suggest the importance of residues Glu-4, Gln-14, Glu-21, and Asp-51. The first 3 of these residues define a face of acyl carrier protein that includes the beginning of the loop region, residues 16 to 36. Additionally, screening for membrane glucosyltransferase activity in membranes from bacterial species that had acyl carrier proteins that were active with E. coli membranes revealed the presence of glucosyltransferase activity only in the species most closely related to E. coli. Thus, it seems likely that only bacteria from the Proteobacteria subgroup gamma-3 have periplasmic glucans synthesized by the mechanism found in E. coli.

Acyl Carrier Protein↗
PubMed1992

Impact of uncoupling glucose stimulus from secretion on B-cell release and biosynthesis.

We studied the impact of a defined degree of long-term hyperglycemia with or without blockade of attendant insulin release on subsequent B-cell secretory responsiveness and biosynthesis. Nondiabetic rats were infused for 48 h with glucose to produce marked hyperglycemia (21.3 +/- 0.5 mmol/l). Comparable levels of hyperglycemia were upheld when additions were made to this protocol. Hyperglycemia increased plasma insulin 12-fold but depressed glucose (27 mmol/l)-induced insulin secretion in vitro (isolated islets) by 67% compared with saline-infused rats. Addition of diazoxide infusion during hyperglycemia completely inhibited the hyperglycemia-induced rise in plasma insulin but enhanced glucose-induced insulin release in vitro eightfold compared with islets from rats infused with glucose alone. Addition of insulin (2 U/day) to the diazoxide plus hyperglycemia protocol inhibited the secretory response to glucose in vitro by 46% (P less than 0.05). Proinsulin biosynthesis was enhanced by 67% in islets from rats infused with glucose alone; this effect was paralleled by a similar increase in preproinsulin mRNA. Diazoxide in vivo did not affect these stimulatory effects of hyperglycemia on insulin biosynthesis; however, insulin infusion in vivo abolished the hyperglycemia-induced increase in proinsulin biosynthesis. We conclude that impairment by hyperglycemia of glucose-induced insulin secretion occurs concomitant with stimulation of biosynthesis. Uncoupling of glucose stimulus from secretion crucially affects subsequent secretory responsiveness but not biosynthesis. Insulin biosynthesis is depressed by direct or indirect effects of circulating insulin.

Animals↗