Genetic activity of deoxyribonucleic acid in the reconstitution of biosynthetic pathways.
Explore the source record for details and available documents.
Biosynthetic Pathways↗
SEARCH · PubMed Health
Results for “Biosynthetic Pathways”
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Liver receptor homolog-1 stimulates the progesterone biosynthetic pathway during follicle-stimulating hormone-induced granulosa cell differentiation.
FSH-stimulated granulosa cell differentiation is associated with the induction of the LH receptor (LHr) as well as induction of the estrogen and progesterone biosynthetic pathways. Although activation of the cAMP-protein kinase A pathway is sufficient to stimulate progesterone production, additional pathways are required for the induction of the LHr and p450 aromatase. The orphan nuclear receptor, liver receptor homolog-1 (LRH-1), is expressed in granulosa cells and has been shown to synergize with the cAMP signaling system to regulate the gonadal type II aromatase promoter in transient transfection assays. To determine whether LRH-1 can interact with the cAMP pathway in the induction of aromatase and the LHr, we examined the effects of an adenoviral vector that directs the expression of human LRH-1 (Ad-LRH-1) on FSH-stimulated granulosa cell differentiation. Infection of undifferentiated granulosa cells with LRH-1 alone had no effect on estrogen production, progesterone production, or the expression of the LHr. However, combination of FSH stimulation and Ad-LRH-1 infection led to significantly greater progesterone production and increases in mRNA for p450 side-chain cleavage and 3beta-hydroxysteroid dehydrogenase than granulosa cells stimulated by FSH alone. However, infection with Ad-LRH-1 did not stimulate estradiol production or increases in mRNA for p450 aromatase or the LHr above that seen with FSH treatment alone. Moreover, infection with Ad-LRH-1 was able to overcome H-89 inhibition of FSH-stimulated progesterone but not estrogen production. Collectively, these observations support a direct role for LRH-1 in the induction of the progesterone but not the estrogen biosynthetic pathway during granulosa cell differentiation.
8-Bromo Cyclic Adenosine Monophosphate↗
Evidence for the thiamine biosynthetic pathway in higher-plant plastids and its developmental regulation.
Thiamine or vitamin B-1, is an essential constituent of all cells since it is a cofactor for two enzyme complexes involved in the citric acid cycle, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. Thiamine is synthesized by plants, but it is a dietary requirement for humans and other animals. The biosynthetic pathway for thiamine in plants has not been well characterized and none of the enzymes involved have been isolated. Here we report the cloning and characterization of two cDNAs representing members of the maize thi1 gene family encoding an enzyme of the thiamine biosynthetic pathway. This assignment was made based on sequence homology to a yeast thiamine biosynthetic gene and by functional complementation of a yeast strain in which the endogenous gene was inactivated. Using immunoblot analysis, the thi1 gene product was found to be located in a plastid membrane fraction. RNA gel blot analysis of various tissues and developmental stages indicated thi1 expression was differentially regulated in a manner consistent with what is known about thiamine synthesis in plants. This is the first report of cDNAs encoding proteins involved in thiamine biosynthesis for any plant species.
Amino Acid Sequence↗
Ketopremithramycins and ketomithramycins, four new aureolic acid-type compounds obtained upon inactivation of two genes involved in the biosynthesis of the deoxysugar moieties of the antitumor drug mithramycin by Streptomyces argillaceus, reveal novel insights into post-PKS tailoring steps of the mithramycin biosynthetic pathway.
Mithramycin is an aureolic acid-type antimicrobial and antitumor agent produced by Streptomyces argillaceus. Modifying post-polyketide synthase (PKS) tailoring enzymes involved in the production of mithramycin is an effective way of gaining further information regarding the late steps of its biosynthetic pathway. In addition, new "unnatural" natural products of the aureolic acid-type class are likely to be produced. The role of two such post-PKS tailoring enzymes, encoded by mtmC and mtmTIII, was investigated, and four novel aureolic acid class drugs, two premithramycin-type molecules and two mithramycin derivatives, were isolated from mutant strains constructed by insertional gene inactivation of either of these two genes. From data bank comparisons, the corresponding proteins MtmC and MtmTIII were believed to act as a C-methyltransferase involved in the production of the D-mycarose (sugar E) of mithramycin and as a ketoreductase seemingly involved in the biosynthesis of the mithramycin aglycon, respectively. However, gene inactivation and analysis of the accumulated products revealed that both genes encode enzymes participating in the biosynthesis of the D-mycarose building block. Furthermore, the inactivation of MtmC seems to affect the ketoreductase responsible for 4-ketoreduction of sugar C, a D-olivose. Instead of obtaining premithramycin and mithramycin derivatives with a modified E-sugar upon inactivation of mtmC, compounds were obtained that completely lack the E-sugar moiety and that possess an unexpected 4-ketosugar moiety instead of the D-olivose at the beginning of the lower deoxysaccharide chain. The inactivation of mtmTIII led to the accumulation of 4E-ketomithramycin, showing that this ketoreductase is responsible for the 4-ketoreduction of the D-mycarose moiety. The new compounds of the mutant strains, 4A-ketopremithramycin A2, 4A-keto-9-demethylpremithramycin A2, 4C-keto-demycarosylmithramycin, and 4E-ketomithramycin, indicate surprising substrate flexibility of post-PKS enzymes of the mithramycin biosynthetic pathway. Although the glycosyltransferase responsible for the attachment of D-mycarose cannot transfer the unmethylated sugar to the existing lower disaccharide chain, it can transfer the 4-ketoform of sugar E. In addition, the glycosyltransferase MtmGIV, which is responsible for the linkage of sugar C, is also able to transfer an activated 4-ketosugar. The oxygenase MtmOIV, normally responsible for the oxidative cleavage of the tetracyclic premithramycin B into the tricyclic immediate precursor of mithramycin, can act on a substrate analogue with a modified or even incomplete trisaccharide chain. The same is true for glycosyltransferases MtmGI and MtmGII, both of which partake in the formation and attachment of the A-B disaccharide in mithramycin.
Antibiotics, Antineoplastic↗
Four major transcriptional responses in the methionine/threonine biosynthetic pathway of Saccharomyces cerevisiae.
Genes encoding enzymes in the threonine/methionine biosynthetic pathway were cloned and used to investigate their transcriptional response to signals known to affect gene expression on the basis of enzyme specific-activities. Four major responses were evident: strong repression by methionine of MET3, MET5 and MET14, as previously described for MET3, MET2 and MET25; weak repression by methionine of MET6; weak stimulation by methionine but no response to threonine was seen for THR1, HOM2 and HOM3; no response to any of the signals tested, for HOM6 and MES1. In a BOR3 mutant, THR1, HOM2 and HOM3 mRNA levels were increased slightly. The stimulation of transcription by methionine for HOM2, HOM3 and THR1 is mediated by the GCN4 gene product and hence these genes are under the general amino acid control. In addition to the strong repression by methionine, MET5 is also regulated by the general control.
Blotting, Northern↗
Cloning of the late genes in the ergosterol biosynthetic pathway of Saccharomyces cerevisiae--a review.
Research on the ergosterol biosynthetic pathway in fungi has focused on the identification of the specific sterol structure required for normal membrane structure and function and for completion of the cell cycle. The pathway and its end product are also the targets for a number of antifungal drugs. Identification of essential steps in ergo-sterol biosynthesis could provide new targets for the development of novel therapeutic agents. Nine of the eleven genes in the portion of the pathway committed exclusively to ergosterol biosynthesis have been cloned, and their essentiality for aerobic growth has been determined. The first three genes, ERG9 (squalene synthase), ERG1 (squalene epoxidase), and ERG7 (lanosterol synthase), have been cloned and found to be essential for aerobic viability since their absence would result in the cell being unable to synthesize a sterol molecule. The remaining eight genes encode enzymes which metabolize the first sterol, lanosterol, to ultimately form ergosterol. The two earliest genes, ERG11 (lanosterol demethylase) and ERG24 (C-14 reductase), have been cloned and found to be essential for aerobic growth but are suppressed by mutations in the C-5 desaturase (ERG3) gene and fen1 and fen2 mutations, respectively. The remaining cloned genes, ERG6 (C-24 methylase), ERG2 (D8AE7 isomerase), ERG3 (C-5 desaturase), and ERG4 (C-24(28) reductase), have been found to be nonessential. The remaining genes not yet cloned are the C-4 demethylase and the C-22 desaturase (ERG5).
Cloning, Molecular↗
Phosphorylation by Ca2+/calmodulin-dependent protein kinase II and protein kinase C of sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin.
Sepiapterin reductase, the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin, was stoichiometrically phosphorylated by Ca2+/calmodulin-dependent protein kinase II and protein kinase C (Ca2+/phospholipid-dependent protein kinase) in vitro. Maximal incorporation of phosphate into the enzyme subunit by these was 3.05 +/- 0.05 (n = 4) and 0.74 +/- 0.03 (n = 5) 32P mol per mol enzyme subunit, respectively. The enzyme was not phosphorylated by cyclic nucleotide-dependent protein kinase of either the cAMP-dependent or cGMP-dependent type in this study. Dihydropteridine reductase, another enzyme working in direct supply of tetrahydrobiopterin, was also a good substrate for Ca2+/calmodulin-dependent protein kinase II. Phosphorylation of sepiapterin reductase by these protein kinases modified the kinetic properties of the enzyme. It is likely that these multifunctional Ca(2+)-activated protein kinases may play a role in the regulation of the physiological function of the BH4-generating enzymes in vivo, as was previously found in the case of BH4-requiring enzymes.
Alcohol Oxidoreductases↗
Biosynthetic pathway of 19-noraldosterone in isolated rat glomerulosa cells.
The biosynthetic pathway of 19-noraldosterone (19-noraldo) in isolated rat glomerulosa cells (GC) and fasciculata-reticular cells (FC) was studied by analyzing [14C]pregnenolone metabolism using HPLC and quantification by specific RIA. In GCs, 18,19-dihydroxycorticosterone was detected after 15 min incubation with [14C]pregnenolone, 18-hydroxy-19-norcorticosterone was detected after 30 min and 19-noraldo was detected after 45 min before the appearance of an aldosterone peak. These three mineralocorticoids were not detected in FCs. The results demonstrate that 19-noraldo is synthesized in GCs and then undergoes further metabolism.
18-Hydroxycorticosterone↗
Neopikromycin and novapikromycin from the pikromycin biosynthetic pathway of Streptomyces venezuelae.
Two new macrolides from the pikromycin biosynthetic pathway of Streptomyces venezuelae, neopikromycin (9) and novapikromycin (10), were identified and structurally characterized through mass spectrometry and NMR spectroscopy. The established structures showed that 9 and 10 have hydroxyl groups at C-14 (9) and at both C-12 and C-14 (10), on the basis of a comparison with narbomycin (7). The purified PikC cytochrome P450 monooxygenase catalyzes the in vitro hydroxylation of 7 and pikromycin (8) to yield 9 and 10, respectively, thus expanding the substrate- and regio-flexibility of this enzyme.
Anti-Bacterial Agents↗
Biosynthetic pathways in Mycobacterium leprae.
The scope of this paper is biosynthetic pathways which are found generally in living organisms. Many are present in M. leprae but some are lacking, perhaps reflecting the parasitic mode of existence of this microbe. The biosynthesis of nucleotides (the immediate precursors of nucleic acids) and fatty acids (intermediates in the biosynthesis of most if not all mycobacterial lipids) has been studied in detail in M. leprae. For the former, M. leprae appears incapable of synthesising its own purines de novo and thus depends upon the host for a source of the purine ring. It can synthesise pyrimidines, though enzymes for this activity are inhibited in the M. leprae organisms obtained from host tissue. For the latter, M. leprae is capable of fatty acid biosynthesis de novo, probably at a repressed level. However, a key enzyme, phosphotransacetylase, appears to be deficient.
DNA, Bacterial↗
Tissue-specific transcriptional regulation of the cholesterol biosynthetic pathway leads to accumulation of testis meiosis-activating sterol (T-MAS).
Lanosterol 14alpha-demethylase (CYP51) produces follicular fluid meiosis-activating sterol (FF-MAS), which is converted further to testis meiosis-activating sterol (T-MAS). MAS are intermediates in the cholesterol biosynthetic pathway, with the ability to trigger resumption of oocyte meiosis in vitro. In contrast to the liver, where pre- and post-MAS genes are upregulated coordinately at the level of transcription by a cholesterol feedback mechanism through sterol regulatory element-binding proteins (SREBP), regulation differs in the testis. Genes encoding pre-MAS enzymes [HMG-CoA synthase (SYN), HMG-CoA reductase (RED), farnesyl diphosphate synthase (FPP), squalene synthase (SS), and CYP51] are upregulated during sexual development of the testis, although not all genes are turned on at the same time. Furthermore, two post-MAS genes, C-4 sterol methyl oxidase and sterol Delta(7)-reductase, are expressed at low levels and are not upregulated either in rat or human. This transcriptional discrepancy seems to be SREBP independent. Besides cAMP/cAMP-responsive element modulator, other unknown transcription factors control expression of individual cholesterogenic genes during spermatogenesis. HPLC analysis shows an 8-fold increase in T-MAS during development of rat testis whereas MAS is barely detectable in livers of the same animals. We propose that the lack of a coordinate transcriptional control over the cholesterol biosynthetic pathway contributes importantly to overproduction of the signaling sterol T-MAS in testis.
Animals↗
Regulation of cholesterol biosynthetic pathway in patients with the Smith-Lemli-Opitz syndrome.
The Smith-Lemli-Opitz syndrome (SLOS) is a recessively inherited birth disorder caused by a defect in 7-dehydrocholesterol (3beta-hydroxysteroid) delta7-reductase, the final enzyme in cholesterol biosynthesis. To investigate in vivo regulation of the cholesterol biosynthetic pathway in SLOS, we measured hepatic microsomal sterol concentrations and activities of several key enzymes in the pathway, including HMG-CoA synthase, HMG-CoA reductase, squalene synthase and 7-dehydrocholesterol delta7-reductase in liver specimens from a patient with SLOS and 11 controls. Hepatic microsomal 7-dehydrocholesterol delta7-reductase activity in the patient was less than 1% of the control mean, and decreased cholesterol concentration and markedly increased 7- and 8-dehydrocholesterol concentrations were observed in the patient's microsomes. HMG-CoA synthase and squalene synthase activities in the patient were upregulated to 149% and 532%, respectively, while the activity of HMG-CoA reductase, the rate-limiting enzyme in the pathway, was reduced to 39% of the control mean. Downregulation of HMG-CoA reductase activity in SLOS was supported by measuring plasma levels of mevalonic acid, the immediate product of HMG-CoA reductase. The levels in SLOS patients (n = 9) were significantly low compared with age-matched controls (n = 8) (12+/-2 vs 28 + 6nmol/L, p < 0.05). These results suggest that in most SLOS patients in vivo HMG-CoA reductase is not stimulated in spite of blocked cholesterol biosynthetic pathway and reduced plasma and hepatic cholesterol concentrations.
Case-Control Studies↗
Channeling of carbamoyl phosphate to the pyrimidine and arginine biosynthetic pathways in the deep sea hyperthermophilic archaeon Pyrococcus abyssi.
The kinetics of the coupled reactions between carbamoyl-phosphate synthetase (CPSase) and both aspartate transcarbamoylase (ATCase) and ornithine transcarbamoylase (OTCase) from the deep sea hyperthermophilic archaeon Pyrococcus abyssi demonstrate the existence of carbamoyl phosphate channeling in both the pyrimidine and arginine biosynthetic pathways. Isotopic dilution experiments and coupled reaction kinetics analyzed within the context of the formalism proposed by Ovádi et al. (Ovádi, J., Tompa, P., Vertessy, B., Orosz, F., Keleti, T., and Welch, G. R. (1989) Biochem. J. 257, 187-190) are consistent with a partial channeling of the intermediate at 37 degrees C, but channeling efficiency increases dramatically at elevated temperatures. There is no preferential partitioning of carbamoyl phosphate between the arginine and pyrimidine biosynthetic pathways. Gel filtration chromatography at high and low temperature and in the presence and absence of substrates did not reveal stable complexes between P. abyssi CPSase and either ATCase or OTCase. Thus, channeling must occur during the dynamic association of coupled enzymes pairs. The interaction of CPSase-ATCase was further demonstrated by the unexpectedly weak inhibition of the coupled reaction by the bisubstrate analog, N-(phosphonacetyl)-L-aspartate (PALA). The anomalous effect of PALA suggests that, in the coupled reaction, the effective concentration of carbamoyl phosphate in the vicinity of the ATCase active site is 96-fold higher than the concentration in the bulk phase. Channeling probably plays an essential role in protecting this very unstable intermediate of metabolic pathways performing at extreme temperatures.
Adaptation, Biological↗
The biosynthetic pathway of vitamin C in higher plants.
Vitamin C (L-ascorbic acid) has important antioxidant and metabolic functions in both plants and animals, but humans, and a few other animal species, have lost the capacity to synthesize it. Plant-derived ascorbate is thus the major source of vitamin C in the human diet. Although the biosynthetic pathway of L-ascorbic acid in animals is well understood, the plant pathway has remained unknown-one of the few primary plant metabolic pathways for which this is the case. L-ascorbate is abundant in plants (found at concentrations of 1-5 mM in leaves and 25 mM in chloroplasts) and may have roles in photosynthesis and transmembrane electron transport. We found that D-mannose and L-galactose are efficient precursors for ascorbate synthesis and are interconverted by GDP-D-mannose-3,5-epimerase. We have identified an enzyme in pea and Arabidopsis thaliana, L-galactose dehydrogenase, that catalyses oxidation of L-galactose to L-galactono-1,4-lactone. We propose an ascorbate biosynthesis pathway involving GDP-D-mannose, GDP-L-galactose, L-galactose and L-galactono-1,4-lactone, and have synthesized ascorbate from GDP-D-mannose by way of these intermediates in vitro. The definition of this biosynthetic pathway should allow engineering of plants for increased ascorbate production, thus increasing their nutritional value and stress tolerance.
Arabidopsis↗
Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.
The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.
Amino Acid Sequence↗
Fine tuning therapeutic targeting of the sphingolipid biosynthetic pathway to treat atherosclerosis.
The accumulation of sphingolipids, including sphingomyelin and glycosphingolipids, in atherosclerotic lesions is well known. Plasma sphingomyelin concentration is correlated with atherosclerosis development and is an independent predictor of coronary artery disease. Similarly, plasma glycosphingolipid levels are increased in conditions associated with atherosclerosis risk. Recent studies have focused on understanding the mechanisms by which specific intermediates and end-products of the sphingolipid biosynthetic pathway, such as sphingomyelin, glycosphingolipids, ceramide and sphingosine-1-phosphate may modulate vascular biology and atherosclerosis. Here we focus on recent work indicating that pharmacological modulation of the sphingolipid biosynthetic pathway could offer a novel treatment for atherosclerosis or, at the very least, provide mechanistic insights concerning the etiology of this disease which is the major cause of death in developed countries.
Animals↗
The discovery of nature's biosynthetic pathways.
Uro'gen-III is a key intermediate on the biosynthetic pathways to the vitally important natural pigments haem, chlorophyll and the cytochromes. How the unexpected structure of uro'gen-III is synthesized by living things has long been a major puzzle. Studies based on 13C-labelling are described which show a) that a single intramolecular rearrangement occurs and b) that this step occurs after the open-chain linear tetrapyrrole system has been built. A second study involves stereospecific labelling with deuterium and tritium to elucidate the absolute stereochemistry of the enzymic reaction sequence which produces the vinyl groups of haem. The third and last section of the lecture is focussed on the biosynthetic intermediates lying between uro'gen-III and cobyrinic acid on the pathway to vitamin B12. An octacarboxylic isobacteriochlorin is isolated from a vitamin B12-producing organism and this is shown to be identical with sirohydrochlorin, previously obtained by Kamin and Siegel as the metal-free prosthetic group of certain sulphite-reducing bacteria. The structure and absolute stereochemistry of sirohydrochlorin are studied and comment is made on the evolutionary interest of these findings.
Chlorophyll↗
Biosynthetic pathway leading to gentamicin C2b.
Incubation of Micromonospora purpurea SC 1210 (NRRL 5467) with L-[methyl-14C]methionine yielded [methyl-14C]gentamicin A and methyl-14C-labeled antibiotic JI-20A in a molar radioactivity ratio of 9:2. We did not isolate methyl-14C-labeled gentamicin X2, which was expected as an intermediate based on the biosynthetic pathways proposed by others. Addition of methyl-14C-labeled antibiotic JI-20A to M. purpurea SC 1124 (NRRL 8102) yielded [methyl-14C]-gentamicin C1a and [methyl-14C]gentamicin C2b in variable molar radioactivity ratios. These data do not support the biosynthetic pathway leading to gentamicin C2b proposed by Testa and Tilley.
Fermentation↗