[Use of yeast mutants for examining common biosynthetic pathways of dolichols, sterols and ubiquinones].
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Dolichols↗
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Results for “Biosynthetic Pathways”
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Directed evolution of biosynthetic pathways. Recruitment of cysteine thioethers for constructing the cell wall of Escherichia coli.
We report that expansion of thioether biosynthesis in Escherichia coli generates sulfur-containing amino acids that can replace meso-diaminopimelate, the essential amino acid used for cross-linking the cell wall. This was accomplished by jointly overexpressing the metB gene coding for L-cystathionine gamma-synthase and disrupting the metC gene, whose product, L-cystathionine beta-lyase, is responsible for the destruction of L-cystathionine and other L-cysteine thioethers. As a result, meso-lanthionine and L-allo-cystathionine were produced endogenously and incorporated in the peptidoglycan, thereby enabling E. coli strains auxotrophic for diaminopimelate to grow in its absence. Thus, current techniques of metabolic engineering can be applied to evolving the chemical constitution of living cells beyond its present state.
Alanine↗
Inhibition of the pyrimidine biosynthetic pathway with S-8660, an analogue of brequinar sodium, prolongs cardiac allograft survival in rats.
The compound S-8660 is a member of a family of antiproliferative drugs that act on de novo pyrimidine synthesis through selective inhibition of the mitochondrial enzyme dihydroorotate dehydrogenase. S-8660 is highly effective in preventing the development of delayed-type hypersensitivity in mice and in suppressing human mixed-lymphocyte responses. We have tested its ability to prevent cardiac allograft rejection in the ACI (RT1a) to Lewis (RT1(1)) rat strain combination, based on the immunosuppressive activity of this compound and its similarity to another member of this group, brequinar sodium. Daily oral administration of the drug (5 to 20 mg/kg) was begun 2 days before transplantation and extended for periods of time up to 30 days after graft placement. Control grafts were promptly rejected (median survival time, 7.0 +/- 0.5 days). Administration of S-8660 was effective in extending graft survival in a dose-dependent fashion. The efficacy of S-8660 could be improved with a high initial concentration of the drug, followed by a reduction ("tapering") in the level of drug administration (median survival time, 32.0 +/- 4.6 days) or by use in combination with cyclosporine. The differences in the mode of action of S-8660, when compared to cyclosporine or FK 506, suggest that the disruption of de novo pyrimidine synthesis may be an effective and safe addition to a polytherapeutic approach for the prevention of allograft rejection in clinical transplantation.
Animals↗
[Carotenoid biosynthetic pathway elucidated at the gene level].
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Carotenoids↗
Fungal glycoproteins and their biosynthetic pathway as potential targets for antifungal agents.
The yeast cell wall as a good antifungal target is discussed in general. More specifically the reaction, catalyzed by Dol-P-Man: protein O-D-mannosyltransferase is proposed as a new potential target. Six genes responsible for this endoplasmic reticulum-localized reaction have been cloned and characterized so far. Triple disruptions of these genes are either lethal or the corresponding cells have to be osmotically stabilized to survive. No inhibitors of this reaction are as yet known.
Antifungal Agents↗
Cytochrome P450 and its interactions with the heme biosynthetic pathway.
An important factor in disruption of hepatic heme biosynthesis by porphyrinogenic drugs appears to be interaction with one or several cytochrome P450 isozymes. Clarification of the nature of the interaction between porphyrinogenic drugs and cytochrome P450 isozymes, as well as identification of the isozymes involved, will be helpful in extrapolating the results of animal experimentation to humans. Administration of griseofulvin to mice results in accumulation in the liver of two N-alkylated protoporphyrins (PPs). The major N-alkyl PP, N-griseofulvin PP, which is devoid of ferrochelatase-inhibitory activity, was shown to be the precursor of N-methyl PP, which is a potent ferrochelatase inhibitor. N-Griseofulvin PP was present predominantly as the Nc regioisomer rather than the anticipated NA regioisomer. Progesterone (PG) 6 beta-hydroxylase and androstenedione (AD) 6 beta-hydroxylase, diagnostic markers for cytochrome P450 3A1/2 activity in rat liver, were identified in chick embryo liver. The in ovo administration of 3,5-diethoxycarbonyl-1,4-dihydro-2,6-dimethyl-4-ethylpyridine (4-ethyl DDC) and 3-[2-(2,4,6-trimethylphenyl)thioethyl]-4-methylsydnone (TTMS) caused inactivation of chick embryo hepatic PG and AD 6 beta-hydroxylases. Ascorbate was shown to inhibit uroporphyrin accumulation in cultures of chick embryos treated with porphyrinogenic drugs and in the livers of an ascorbate-requiring rat strain treated with a porphyrinogenic combination of chemicals. Ascorbate appears to act by inhibiting oxidation of uroporphyrinogen to uroporphyrin. Avian hepatocytes were cultured in 48-well plates and directly assayed within the wells for the activity of 7-ethoxyresorufin O-deethylation (EROD), and for porphyrin and protein concentration by a fluorescence plate reader. Uroporphyrin was the main porphyrin to accumulate in response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in chick embryos, pheasants, ducks, and herring gulls; heptacarboxylated porphyrin predominated in turkey hepatocytes. The EROD-inducing potential of complex mixtures of polyhalogenated aromatic hydrocarbons from herring gull eggs was assessed and compared with that of TCDD. A nuclear heme pool appears to regulate the transcription of the cytochrome P450 2B1/2B2 genes in rat liver, by modulating binding of a transcription factor(s) that appears to be involved in the transcriptional activation by phenobarbitone.
Animals↗
Proceedings: Coordinate control of nucleotide biosynthetic pathways.
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Animals↗
Biosynthesis of tunicamycin and metabolic origin of the 11-carbon dialdose sugar, tunicamine.
Tunicamycin is a reversible inhibitor of polyprenol-phosphate: N-acetylhexosamine-1-phosphate translocases and is produced by several Streptomyces species. We have examined tunicamycin biosynthesis, an important but poorly characterized biosynthetic pathway. Biosynthetic precursors have been identified by incorporating radioactive and stable isotopes, and by determining the labeling pattern using electrospray ionization-collision induced dissociation-mass spectrometry (ESI-CID-MS), and proton, deuterium, and C-13 nuclear magnetic resonance (NMR) spectroscopy. Preparation and analysis of [uracil-5-(2)H]-labeled tunicamycin established the complete ESI-CID-MS fragmentation pathway for the major components of the tunicamycin complex. Competitive metabolic experiments indicate that 7 deuteriums incorporate into tunicamycin from [6,6'-(2)H,(2)H]-labeled D-glucose, 6 of which arise from D-GlcNAc and 1 from uridine and/or D-ribose. Inverse correlation NMR experiments (heteronuclear single-quantum coherence (HSQC)) of (13)C-labeled tunicamycin enriched from D-[1-(13)C]glucose suggest that the unique tunicamine 11-carbon dialdose sugar backbone arises from a 5-carbon furanose precursor derived from uridine and a 6-carbon N-acetylamino-pyranose precursor derived from UDP-D-N-acetylglucosamine. The equivalent incorporation of (13)C into both the alpha-1" and beta-11' anomeric carbons of tunicamycin supports a direct biosynthesis via 6-carbon metabolism. It also indicates that the tunicamine motif and the alpha-1"-linked GlcNAc residue are both derived from the same metabolic pool of UDP-GlcNAc, without significant differential metabolic processing. A biosynthetic pathway is therefore proposed for tunicamycin for the first time: an initial formation of the 11-carbon tunicamine sugar motif from uridine and UDP-GlcNAc via uridine-5'-aldehyde and UDP-4-keto-6-ene-N-acetylhexosamine, respectively, and subsequent formation of the anomeric-to-anomeric alpha, beta-1",11'-glycosidic bond.
Carbohydrate Sequence↗
In silico assessment of gene function involved in cysteine biosynthesis in Arabidopsis: expression analysis of multiple isoforms of serine acetyltransferase.
In plants, the inorganic sulfur is first fixed into cysteine by the cysteine biosynthetic pathway. This biosynthetic pathway of cysteine involves several enzymatic reactions. In Arabidopsis thaliana, multiple isoforms seem to participate in each enzymatic step for cysteine biosynthesis. To obtain more insights on the specific role of each isoform involved in the cysteine biosynthesis, in silico analysis of these isoforms using Arabidopsis expressed sequence tags (EST) database was carried out. This EST database analysis revealed distinct population distribution of ESTs among multiple isoforms, suggesting that each isoform has its particular expression pattern, presumably associated with its specific role in cysteine biosynthesis. As another in silico analysis, co-expression analysis of genes involved in sulfur metabolism in Arabidopsis was performed using a public transcriptome database of DNA microarrays. This co-expression analysis also suggested specific function and co-regulation of some isoform genes for cysteine biosynthesis by consideration on the clustering of co-expressed genes. From the results of sensitivity to feedback regulation, subcellular localization and expression of mRNA analyses, each serine acetyltransferase (SATase) isoform seems to have its specific role for cysteine biosynthesis. Similar expression patterns were observed between the experimental results of expression data for SATase isoforms and the in silico results of "digital northern" analysis using EST database.
Arabidopsis↗
Biosynthesis of TDP-l-mycarose: the specificity of a single enzyme governs the outcome of the pathway.
The biosynthetic pathway for l-mycarose, a 2,6-dideoxy-3-methylhexose found in the antibiotic tylosin produced by Streptomyces fradiae, has been studied with only two genes in the gene cluster, tylK and tylC2, remaining to be characterized. To verify the postulated functions of these two genes and to establish the sequence of events in the pathway, functional analyses of the proteins encoded by tylK and tylC2 were carried out. The results clearly show that TylK functions as the 5-epimerase and TylC2 functions as the 4-ketoreductase. However, both enzymes exhibit relaxed substrate specificity such that the production of TDP-l-mycarose as the sole product of the pathway depends on the absolute specificity of TylC3, a methyltransferase involved in an early step in the pathway. Exploitation of the promiscuous substrate specificity in the late steps of the mycarose pathway could produce new analogues of tylosin as well as other antibiotics containing this sugar.
Anti-Bacterial Agents↗
Biosynthetic studies of aridicin antibiotics. I. Labeling patterns and overall pathways.
Biosynthetic feeding experiments with 14C and 13C-labeled precursors in Kibdelosporangium aridum have established the biosynthetic origins of the heptapeptide aglycone of the aridicin antibiotics, and the tentative sequence of the later stage biosynthetic transformations. The aglycone moiety has been found to be derived from tyrosine, sodium acetate and L-methionine. It is suggested that the preformed aglycone is first mannosylated and then followed by the attachment of the glycolipid. The sugar oxidation to the glucuronic acid level was found to take place as a terminal step of the biosynthesis.
Acetates↗
Crystallographic analysis of the reaction pathway of Zoogloea ramigera biosynthetic thiolase.
Biosynthetic thiolases catalyze the biological Claisen condensation of two acetyl-CoA molecules to form acetoacetyl-CoA. This is one of the fundamental categories of carbon skeletal assembly patterns in biological systems and is the first step in many biosynthetic pathways including those which generate cholesterol, steroid hormones and ketone body energy storage molecules. High resolution crystal structures of the tetrameric biosynthetic thiolase from Zoogloea ramigera were determined (i) in the absence of active site ligands, (ii) in the presence of CoA, and (iii) from protein crystals which were flash frozen after a short soak with acetyl-CoA, the enzyme's substrate in the biosynthetic reaction. In the latter structure, a reaction intermediate was trapped: the enzyme was found to be acetylated at Cys89 and a molecule of acetyl-CoA was bound in the active site pocket. A comparison of the three new structures and the two previously published thiolase structures reveals that small adjustments in the conformation of the acetylated Cys89 side-chain allow CoA and acetyl-CoA to adopt identical modes of binding. The proximity of the acetyl moiety of acetyl-CoA to the sulfur atom of Cys378 supports the hypothesis that Cys378 is important for proton exchange in both steps of the reaction. The thioester oxygen atom of the acetylated enzyme points into an oxyanion hole formed by the nitrogen atoms of Cys89 and Gly380, thus facilitating the condensation reaction. The interaction between the thioester oxygen atom of acetyl-CoA and His348 assists the condensation step of catalysis by stabilizing a negative charge on the thioester oxygen atom. Our structure of acetyl-CoA bound to thiolase also highlights the importance in catalysis of a hydrogen bonding network between Cys89 and Cys378, which includes the thioester oxygen atom of acetyl-CoA, and extends from the catalytic site through the enzyme to the opposite molecular surface. This hydrogen bonding network is different in yeast degradative thiolase, indicating that the catalytic properties of each enzyme may be modulated by differences in their hydrogen bonding networks.
Acetyl Coenzyme A↗
Changes of sterol metabolism during 3-methylcholanthrene-induced mouse skin carcinogenesis.
Changes of sterol metabolism in mouse skin treated with a chemical carcinogen were studied by thin layer and gas-liquid chromatographic analyses, and it was found that some sterols disappeared from the skin. These sterols were identified as members of one of the cholesterol biosynthetic pathways, which proceeds through intermediates with saturated side chains. Furthermore, based on the results of an experiment performed with animals that had been treated with a metabolic inhibitor, azacholesterol, it is suggested that the disappearance of these sterols may be due to enzyme repression instead of increased activity of the pathway. The other biosynthetic pathway for cholesterol synthesis, which proceeds through intermediates with unsaturated side chains, was rather stimulated by the application of this chemical carcinogen to mouse skin. This change in sterol metabolism may be intimately related to the process of chemical carcinogenesis of mouse skin.
Animals↗
Structure, biosynthesis, and function of glycosylphosphatidylinositols.
The last few years have witnessed an explosion in our knowledge of GPI membrane anchors and related glycolipids and molecules where structure details are available, as illustrated in Figure 2. There is now sufficient information on a handful of these molecules to allow a detailed comparison of their chemical structures. Despite a common structural theme, i.e., the presence of mannoglucosaminyl-PI, a great deal of diversity exists in both the glycan structures and the glycerol-linked aliphatic substituents. The complexities of the structures clearly show that a multitechnique approach is required in the elucidation of their structures. The anticipated publication of more structures from a wider range of organisms may reveal even greater diversity, as well as suggesting possible biosynthetic pathways. The details of a potential biosynthetic pathway in T. brucei are becoming apparent, but confirmation of its importance awaits the isolation and characterization of the enzymes involved. Leishmania, in which LPG, GPIs, GIPLs, and GPI membrane anchors are produced, may also provide an interesting system for biosynthetic studies. The recent description of a GPI biosynthetic system in yeast may provide the crucial breakthroughs necessary in unraveling the enzymes and sugar donors involved in the biosynthetic pathway and possibly the role of the GPI membrane anchor in the function of proteins containing these moieties. Knowledge of the solution structure (conformation), in addition to the complete chemical structure, of the T. brucei VSG anchor has led to speculation that the glycan fulfills a space-filling role in the VSG coat. Many other possible roles of GPI membrane anchors have been suggested, including the shedding and turnover of membrane proteins, signal transduction, and intracellular targeting. Nevertheless, the only function of GPIs that we can so far be certain of is that they anchor proteins or polysaccharide to a membrane. Regardless of the roles GPIs may or may not ultimately be shown to play, the fact that such a widely occurring structure has only recently been characterized serves as a reminder of the incompleteness of our knowledge of biological phenomena and the constant possibility of finding novel molecules in obvious places.
Animals↗
Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (review).
Glycosylphosphatidylinositols (GPIs) represent an abundant and ubiquitous class of eukaryotic glycolipids. Although these structures were originally discovered in the form of GPI-anchored cell surface glycoproteins, it is becoming increasingly clear that a significant proportion of the GPI synthetic output of a cell is not directed to protein anchoring. Indeed, pools of non-protein-linked GPIs can approach 10(7) molecules per cell in some cell types, especially the protozoa, with a large proportion of these molecules being displayed at the cell surface. Recent studies which form the subject of this review indicate that there is (a) considerable diversity in the range of structural modifications found on GPI glycolipids within and between species and cell types, (b) complexity in the topological arrangement of the GPI biosynthetic pathway in the endoplasmic reticulum, and (c) spatial restriction of the biosynthetic pathway within the endoplasmic reticulum. Furthermore, consistent with additional functional roles for these lipids beyond serving as protein anchor precursors, products of the GPI biosynthetic pathway appear to be widely distributed in the cellular endomembrane system. These studies indicate that there is still much to learn about the organization of glycolipid biosynthetic pathways in eukaryotic cells, the nature and subcellular distribution of the lipid products of these pathways, and the function of these lipids within cells.
Animals↗
Design of molecular control mechanisms and the demand for gene expression.
Regulation by a repressor protein is the mechanism selected when, in the organism's natural environment, there is low demand for expression of the regulated structural genes. Regulation by an activator protein is selected when there is high demand for expression of the regulated structural genes. These general conclusions are useful in relating physiological function to underlying molecular determinants in a wide variety of systems that includes repressible biosynthetic pathways, inducible biosynthetic enzymes, inducible drug resistance, and prophage induction, as well as inducible catabolic pathways, for which a special case of this prediction previously was reported [Savageau, M. A. (1974) Proc. Natl. Acad. Sci. USA 71, 2453-2455].
Bacteria↗
Brassinosteroids are inherently biosynthesized in the primary roots of maize, Zea mays L.
GC-MS analysis revealed that primary roots of maize contain 6-deoxocathasterone, 6-deoxoteasterone and 6-deoxotyphasterol. These brassinosteroids, and the previously identified campesterol, campestanol, 6-deoxocastasterone and castasterone, in the roots are members of a biosynthetic pathway to castasterone, namely the late C-6 oxidation pathway, suggesting that its biosynthetic pathway is operative in the roots. To verify this, a cell-free enzyme extract was prepared from maize roots, and enzymatic conversions from campesterol to castasterone through the aforementioned sterols and brassinosteroids were examined. The presence for the biosynthetic sequences, campesterol-->24-methylcholest-4-en-3beta-ol-->24-methylcholest-4-en-3-one-->24-methylcholest-5 alpha-cholestan-3-one-->campestanol and 6-deoxoteasterone-->6-deoxo-3-dehydroteasterone-->6-deoxotyphasterol-->6-deoxocastasterone-->castasterone were demonstrated. These results indicate that maize roots contain a complete set of enzymes involved in the late C-6 oxidation pathway, thereby demonstrating that endogenous brassinosteroids are biosynthesized in the roots.
Models, Chemical↗
Biosynthesis of chloroplastidic and extrachloroplastidic terpenoids in liverwort cultured cells: 13C serine as a probe of terpene biosynthesis via mevalonate and non-mevalonate pathways.
Two terpenoid biosynthetic pathways, the mevalonate and non-mevalonate (glyceraldehyde phosphate-pyruvate) routes, were examined by feeding (13)C-labeled serines ([1-(13)C]- and [3-(13)C]-) to the cultured cells of the liverwort, Heteroscyphus planus. The labeling patterns observed in the isoprenoid unit of the biosynthetically (13)C-labeled stigmasterol corresponded to those expected from the mevalonate pathway, while those of the phytyl side chain corresponded to those from the non-mevalonate pathway. Thus, serine is a potential probe to determine the origin of terpenoid biosynthesis, in either the mevalonate or non-mevalonate pathway.
Acetylation↗