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PubMed2004

Recombinant Saccharomyces cerevisiae strain triggers acetate production to fuel biosynthetic pathways.

Although the metabolism and physiology of the growth of yeast strains has been extensively studied, many questions remain unanswered when the induced production of a recombinant protein is concerned. This work addresses the production of a Fusarium solani pisi cutinase by a recombinant Saccharomyces cerevisiae strain induced through the use of a galactose promoter. It was observed that whenever the strain needed to activate biosynthetic pathways, either for cutinase synthesis, or for the synthesis of the enzymes required for galactose intake, acetate production occurred. The on-line detection of acetate in the medium might prove useful for the control and the supervision of recombinant protein production processes using yeast. The volumes of acid and base added to control the pH throughout the time course of the cultivations were used to calculate an on-line estimator for acetate concentration.

Acetic Acid↗

PubMed1972

Isotopic study of control of the lysine biosynthetic pathway during sporulation of Bacillus cereus.

The extent of incorporation of aspartate into dipicolinic acid and into various amino compounds was determined in Bacillus cereus at various times before, during, and near the end of synthesis of dipicolinic acid. The purpose of this study was to gain further information on the in vivo control of the biosynthesis of amino acids derived from aspartate. Control of the lysine biosynthetic pathway was of particular interest with regard to sporulation, owing to the important role of diaminopimelate and dipicolinate in the structure of the spore. As synthesis of dipicolinate was initiated, incorporation of carbon derived from aspartate was funneled preferentially into this compound as compared with others of the aspartate group. Incorporation into lysine essentially stopped just before the synthesis of dipicolinate began. This is consistent with the previously observed disappearance at this time of diaminopimelic acid decarboxylase in cell-free extracts. Synthesis of diaminopimelate continued during the time of synthesis of dipicolinate. The previous suggestion that diaminopimelate might exert negative control on one of the enzymes between dihydrodipicolinate and diaminopimelate is thus considered unlikely. The possibility is discussed that synthesis of dipicolinate is favored by an increase in the rate of synthesis of dihydrodipicolinate rather than by a block in its rate of utilization.

Aspartic Acid↗

PubMed2003

Biosynthetic pathways of plastid-derived organelles as potential drug targets against parasitic apicomplexa.

Apicomplexan parasites are a large phylum of unicellular and obligate intracellular organisms of great medical importance. They include the human pathogens Plasmodium spp., the causative agent of malaria, and Toxoplasma gondii, an opportunistic parasite of immunosuppressed individuals and a common cause of congenital disease, together affecting several hundred million people worldwide. The search for new and effective drugs against these pathogens has been boosted during the last years by an unexpected finding. Through molecular and cell biological analysis it was realized that probably most members of this phylum harbor a plastid-like organelle, called the apicoplast, which probably is derived from the engulfment of a red alga in ancient times. Although the apicoplast itself contains a small circular genome, most of the proteome of this organelle is encoded in the nuclear genome, and the proteins are subsequently transported to the apicoplast. It is assumed to contain a number of unique metabolic pathways not found in the vertebrate host, making it an ideal "playground" for those interested in drug targets. Recent reports have shown that the rationale of this approach is valid and that new drugs which are urgently needed especially for plasmodial infections, might be developed in the near future based on these targets. Amongst them are three enzymes of the plant-like fatty acid synthesis machinery and enzymes of the non-mevalonat isoprenoid biosynthesis pathway. From their presence in the apicoplast it can be concluded that fatty acid and lipid biosynthesis seems to be a major function of the apicoplast. Another recently described apicoplast enzyme, ferredoxin-NADP(+)-reductase and its redox partner, ferredoxin, points to another interesting organelle-specific biosynthetic pathway, namely [Fe-S] cluster biosynthesis. In the present review, the fundamental aspects of the apicoplast as drug target will be described, together with the specific pathways and their currently known inhibitors. Furthermore, based on the recent findings potentially new targets will be discussed. A short overview of the presently available high-throughput methods for Apicomplexa to evaluate the potency of new inhibitory substances will also be given.

Animals↗

PubMed1996

The tryptophan biosynthetic pathway of aphid endosymbionts (Buchnera): genetics and evolution of plasmid-associated anthranilate synthase (trpEG) within the aphididae.

The bacterial endosymbionts (Buchnera) from the aphids Rhopalosiphum padi, R. maidis, Schizaphis graminum, and Acyrthosiphon pisum contain the genes for anthranilate synthase (trpEG) on plasmids made up of one or more 3.6-kb units. Anthranilate synthase is the first as well as the rate-limiting enzyme in the tryptophan biosynthetic pathway. The amplification of trpEG on plasmids may result in an increase of enzyme protein and overproduction of this essential amino acid, which is required by the aphid host. The nucleotide sequence of trpEG from endosymbionts of different species of aphids is highly conserved, as is an approximately 500-bp upstream DNA segment which has the characteristics of an origin of replication. Phylogenetic analyses were performed using trpE and trpG from the endosymbionts of these four aphids as well as from the endosymbiont of Schlechtendalia chinensis, in which trpEG occurs on the chromosome. The resulting phylogeny was congruent with trees derived from sequences of two chromosome-located bacterial genes (part of trpB and 16S ribosomal DNA). In turn, trees obtained from plasmid-borne and bacterial chromosome-borne sequences were congruent with the tree resulting from phylogenetic analysis of three aphid mitochondrial regions (portions of the small and large ribosomal DNA subunits, as well as cytochrome oxidase II). Congruence of trees based on genes from host mitochondria and from bacteria adds to previous support for exclusively vertical transmission of the endosymbionts within aphid lineages. Congruence with trees based on plasmid-borne genes supports the origin of the plasmid-borne trpEG from the chromosomal genes of the same lineage and the absence of subsequent plasmid exchange among endosymbionts of different species of aphids.

Amino Acid Sequence↗

PubMed1995

Biosynthetic pathways of glycerol accumulation under salt stress in Aspergillus nidulans.

A culture of Aspergillus nidulans (FGSC 359) was gradually adapted for growth in media containing up to 2 M NaCl or was exposed to a salt shock with 2 M NaCl. The intracellular glycerol level increased by about 7.9-fold in salt-adapted and 2.4-fold in salt-shocked cultures when compared to the unadapted culture. The biosynthetic pathway involved in the accumulation of glycerol was investigated under long-term salt adaptation and short-term salt shock. Glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) was induced 1.4-fold in salt-shocked but not in salt-adapted cultures. An alternate enzymatic pathway involving glycerol dehydrogenase (NADP(+)-dependent) utilizing dihydroxyacetone (DHA) and/or DL-glyceraldehyde (DL-GAD) was induced by NaCl. DHA-dependent glycerol dehydrogenase activity was induced about 6.3-fold in salt-adapted and 1.35-fold in salt-shocked cultures, while DL-GAD-dependent activity was induced about 6.1-fold in salt-adapted and 1.2-fold in salt-shocked cultures. However, the level of glycerol dehydrogenase activity with DL-GAD as substrate was 7% of the DHA-dependent activity. We conclude that a salt-inducible NADP(+)-dependent glycerol dehydrogenase activity electrophoretically indistinguishable from previously described glycerol dehydrogenase I results in glycerol accumulation in salt-stressed A. nidulans.

Aspergillus nidulans↗

PubMed1987

A common origin for enzymes involved in the terminal step of the threonine and tryptophan biosynthetic pathways.

Comparison of the amino acid sequence of Bacillus subtilis threonine synthase with the National Biomedical Research Foundation protein sequence library revealed a statistically significant extent of similarity between the sequence of the tryptophan synthase beta chain from various organisms and that of threonine synthase. This homology in the primary structure of threonine synthase and tryptophan synthase beta chain, which catalyze the last step in the threonine and the tryptophan biosynthetic pathways, respectively, correlates well with some of their catalytic properties and indicates that they have evolved from a common ancestor. The evolutionary relationship between these enzymes supports the hypothesis that primitive enzymes possessed a broad substrate specificity and were active in several metabolic pathways.

Bacillus subtilis↗

PubMed2004

Engineering biosynthetic pathways for deoxysugars: branched-chain sugar pathways and derivatives from the antitumor tetracenomycin.

Sugar biosynthesis cassette genes have been used to construct plasmids directing the biosynthesis of branched-chain deoxysugars: pFL942 (NDP-L-mycarose), pFL947 (NDP-4-deacetyl-L-chromose B), and pFL946/pFL954 (NDP-2,3,4-tridemethyl-L-nogalose). Expression of pFL942 and pFL947 in S. lividans 16F4, which harbors genes for elloramycinone biosynthesis and the flexible ElmGT glycosyltransferase of the elloramycin biosynthetic pathway, led to the formation of two compounds: 8-alpha-L-mycarosyl-elloramycinone and 8-demethyl-8-(4-deacetyl)-alpha-L-chromosyl-tetracenomycin C, respectively. Expression of pFL946 or pFL954 failed to produce detectable amounts of a novel glycosylated tetracenomycin derivative. Formation of these two compounds represents examples of the sugar cosubstrate flexibility of the ElmGT glycosyltransferase. The use of these cassette plasmids also provided insights into the substrate flexibility of deoxysugar biosynthesis enzymes as the C-methyltransferases EryBIII and MtmC, the epimerases OleL and EryBVII, and the 4-ketoreductases EryBIV and OleU.

Antineoplastic Agents↗

PubMed2003

Structure and mechanism of Pseudomonas aeruginosa PhzD, an isochorismatase from the phenazine biosynthetic pathway.

PhzD from Pseudomonas aeruginosa is an isochorismatase involved in phenazine biosynthesis. Phenazines are antimicrobial compounds that provide Pseudomonas with a competitive advantage in certain environments and may be partly responsible for the persistence of Pseudomonas infections. In vivo, PhzD catalyzes the hydrolysis of the vinyl ether functional group of 2-amino-2-deoxyisochorismate, yielding pyruvate and trans-2,3-dihydro-3-hydroxyanthranilic acid, which is then utilized in the phenazine biosynthetic pathway. PhzD also catalyzes hydrolysis of the related vinyl ethers isochorismate, chorismate, and 4-amino-4-deoxychorismate. Here we report the 1.5 A crystal structure of native PhzD, and the 1.6 A structure of the inactive D38A variant in complex with isochorismate. The structures reveal that isochorismate binds to the PhzD active site in a trans-diaxial conformation, and superposition of the structures indicates that the methylene pyruvyl carbon of isochorismate is adjacent to the side chain carboxylate of aspartate 38. The proximity of aspartate 38 to isochorismate and the complete loss of activity resulting from the conversion of aspartate 38 to alanine suggest a mechanism in which the carboxylate acts as a general acid to protonate the substrate, yielding a carbocation/oxocarbonium ion that is then rapidly hydrated to form a hemiketal intermediate, which then decomposes spontaneously to products. The structure of PhzD is remarkably similar to other structures from a subfamily of alpha/beta-hydrolase enzymes that includes pyrazinamidase and N-carbamoylsarcosine amidohydrolase. However, PhzD catalyzes unrelated chemistry and lacks a nucleophilic cysteine found in its close structural relatives. The vinyl ether hydrolysis catalyzed by PhzD represents yet another example of the catalytic diversity seen in the alpha/beta-hydrolase family, whose members are also known to hydrolyze amides, phosphates, phosphonates, epoxides, and C-X bonds.

Catalytic Domain↗

PubMed1992

Biosynthetic pathways of the osmolytes N epsilon-acetyl-beta-lysine, beta-glutamine, and betaine in Methanohalophilus strain FDF1 suggested by nuclear magnetic resonance analyses.

Methanohalophilus strain FDF1 synthesizes beta-glutamine, betaine, and N epsilon-acetyl-beta-lysine as osmoprotective agents when the cells are grown in high external concentrations of NaCl. Nuclear magnetic resonance spectroscopic analyses of 13CH3OH-12CO2 label incorporation by the cells provide information on the biosynthetic pathways of these organic osmolytes. The labeling studies indicate that Methanohalophilus strain FDF1 produces glutamate and beta-glutamine via a partial oxidative Krebs pathway. 13C labeling of betaine is consistent with methylation of glycine generated from serine (via serine hydroxymethyltransferase). The labeling pattern for N epsilon-acetyl-beta-lysine is consistent with the synthesis of its precursor alpha-lysine occurring by the diaminopimelate pathway in these cells.

Betaine↗

PubMed2006

A biosynthetic pathway for anandamide.

The endocannabinoid arachidonoyl ethanolamine (anandamide) is a lipid transmitter synthesized and released "on demand" by neurons in the brain. Anandamide is also generated by macrophages where its endotoxin (LPS)-induced synthesis has been implicated in the hypotension of septic shock and advanced liver cirrhosis. Anandamide can be generated from its membrane precursor, N-arachidonoyl phosphatidylethanolamine (NAPE) through cleavage by a phospholipase D (NAPE-PLD). Here we document a biosynthetic pathway for anandamide in mouse brain and RAW264.7 macrophages that involves the phospholipase C (PLC)-catalyzed cleavage of NAPE to generate a lipid, phosphoanandamide, which is subsequently dephosphorylated by phosphatases, including PTPN22, previously described as a protein tyrosine phosphatase. Bacterial endotoxin (LPS)-induced synthesis of anandamide in macrophages is mediated exclusively by the PLC/phosphatase pathway, which is up-regulated by LPS, whereas NAPE-PLD is down-regulated by LPS and functions as a salvage pathway of anandamide synthesis when the PLC/phosphatase pathway is compromised. Both PTPN22 and endocannabinoids have been implicated in autoimmune diseases, suggesting that the PLC/phosphatase pathway of anandamide synthesis may be a pharmacotherapeutic target.

Animals↗

PubMed2005

Biosynthetic pathway of Pseudomonas aeruginosa 4-hydroxy-2-alkylquinolines.

The role of intercellular communication in the regulation of bacterial multicellular behavior has received widespread attention, and a variety of signal molecules involved in bacterial communication have been discovered. In addition to the N-acyl-homoserine lactones, 4-hydroxy-2-alkylquinolines (HAQs), including the Pseudomonas quinolone signal, have been shown to function as signal molecules in Pseudomonas aeruginosa. In this study we unraveled the biosynthetic pathway of HAQs using feeding experiments with isotope-labeled precursors and analysis of extracted HAQs by gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Our results show that the biosynthesis of various HAQ metabolites is directed via a common metabolic pathway involving a "head-to-head" condensation of anthranilic acid and beta-keto fatty acids. Moreover, we provide evidence that the beta-keto-(do)decanoic acids, crucial for the biosynthesis of the heptyl and nonyl derivatives of the 4-hydroxyquinolines in P. aeruginosa, are at least in part derived from a common pool of beta-hydroxy(do)decanoic acids involved in rhamnolipid biosynthesis.

Chromatography, Gas↗

PubMed1969

Bacteriochlorophyll and heme synthesis in Rhodopseudomonas spheroides: possible role of heme in regulation of the branched biosynthetic pathway.

Synthesis of heme, measured by incorporation of iron-59, and of bacteriochlorophyll was studied with wild-type and mutant strains of Rhodopseudomonas spheroides. The wild type formed heme from glycine and succinate at one-fortieth the rate of bacteriochlorophyll under anaerobic-light conditions. Added delta-aminolevulinate stimulated heme synthesis 10-fold without increasing bacteriochlorophyll production. Heme synthesis from glycine and succinate was increased when the magnesium branch of the biosynthetic path was curtailed by mutation or by p-fluorophenylalanine or 8-azaguanine. Synthesis of bacteriochlorophyll by the wild type from glycine and succinate stopped immediately after addition of puromycin, but heme production continued for a period. Porphyrins and other precursors did not appear upon addition of puromycin alone, but simultaneous addition of o-phenanthroline resulted in the accumulation of coproporphyrin. Production of this porphyrin by a mutant strain with impaired ability to form heme was unaffected by puromycin. Heme synthesis from glycine and succinate or from delta-aminolevulinate was decreased by limitation of methionine; it is suggested that coproporphyrin accumulation from glycine and succinate under conditions of methionine deficiency results from relief of feedback inhibition of delta-aminolevulinate synthase by heme. The development of delta-aminolevulinate synthase activity in response to low aeration is prevented by addition of delta-aminolevulinate. This repressive action of the latter is abolished when its conversion to heme is impeded by mutation or by methionine deficiency. It is suggested that heme, the quantitatively minor end product of the branched biosynthetic pathway, may regulate the flow of common intermediates when utilization of protoporphyrin by the magnesium branch is diminished. This regulation may be exerted by feedback inhibition of delta-aminolevulinate synthase and also by repression of enzyme formation.

Amino Acids↗

PubMed1990

The biosynthetic pathway of MHC class II but not class I molecules intersects the endocytic route.

We studied the intracellular traffic and subcellular distribution of MHC class I and class II antigens in comparison with a recycling surface glycoprotein, the transferrin receptor (Tfr), in the human lymphoblastoid cell line JY. No internalization was detectable for class I molecules. Class II molecules were internalized but did not recycle. In contrast, Tfr was found to internalize and recycle. The biosynthetic pathway of class II molecules differ from that of class I molecules in that it shows a delay (1-3 hr) in transport from trans-Golgi to cell surface: here it intersects the endocytic route. Immunoelectron microscopy using anti-MHC antibodies revealed the existence of vesicular structures that were intensely labeled for class II molecules. It is proposed that at this site combination of class II molecules with processed antigen could occur.

B-Lymphocytes↗

PubMed2003

Virus-induced silencing of sterol biosynthetic genes: identification of a Nicotiana tabacum L. obtusifoliol-14alpha-demethylase (CYP51) by genetic manipulation of the sterol biosynthetic pathway in Nicotiana benthamiana L.

Obtusifoliol-14alpha-demethylase (CYP51) is implicated in plant sterol biosynthesis. An Arabidopsis expressed sequence tag encoding a CYP51 was used as a probe to isolate Nicotiana tabacum L. cDNAs. Two types of cDNA clones were identified. Nt CYP51-1 and Nt CYP51-2 shared 97% identity together and around 75% with other plant CYP51s. The function of the encoded enzyme has been demonstrated in planta by manipulating the sterol biosynthetic pathway at the gene level. The endogenous CYP51 of Nicotiana benthamiana was silenced upon inoculation of the plantlets with POTATO VIRUS X::Nt CYP51-1 transcripts. This resulted in the accumulation of obtusifoliol, the substrate of CYP51, and other 14alpha-methyl sterols, with a concomitant growth reduction phenotype. Virus-induced gene silencing was also applied to another steroidogenic enzyme, the Delta7-sterol-C5(6)-desaturase, and this resulted in the accumulation of Delta7-sterols in infected plants instead of the pathway end-products Delta5-sterols.

Cloning, Molecular↗

PubMed1980

Identification of averantin as an aflatoxin B1 precursor: placement in the biosynthetic pathway.

A new blocked mutant of Aspergillus parasiticus produces no detectable aflatoxin B1, but accumulates several polyhydroxyanthraquinones. One of these pigments was identified as averantin. This is the first report of its formation by A. parasiticus. Radiotracer studies with [14C]averantin showed that 15.3% of label from averantin was incorporated into aflatoxin B1. This incorporation was blocked by dichlorvos. With radiotracers and other mutants, averantin was placed after norsolorinic acid and before averufin in the biosynthetic pathway in which the general steps are norsolorinic acid leads to averantin leads to averufin leads to versiconal hemiacetal acetate leads to versicolorin A leads to sterigmatocystin leads to aflatoxin B1.

Aflatoxins↗

PubMed2005

Effects of antirheumatic treatments on the prostaglandin E2 biosynthetic pathway.

OBJECTIVE: Microsomal prostaglandin E synthase 1 (mPGES-1) is up-regulated in experimental arthritis and markedly expressed in synovial tissue biopsy samples from patients with rheumatoid arthritis (RA). This study was carried out to determine the effects of tumor necrosis factor (TNF) blockers and glucocorticoids on mPGES-1 and cyclooxygenase (COX) expression, as well as biosynthesis of PGE(2) in rheumatoid joints. METHODS: In vitro effects of TNF blockers and dexamethasone on the PGE(2) biosynthetic pathway were examined in RA synovial fluid mononuclear cells (SFMCs) by flow cytometry. PGE(2) levels in culture supernatants were measured by enzyme immunoassay. Expression of enzymes responsible for PGE(2) synthesis ex vivo was evaluated by immunohistochemistry in synovial biopsy samples obtained from 18 patients before and after treatment with TNF blockers and from 16 patients before and after intraarticular treatment with glucocorticoids. Double immunofluorescence was performed using antibodies against mPGES-1, COX-1, COX-2, and CD163. RESULTS: Double immunofluorescence revealed that mPGES-1 and COX-2 were colocalized in SFMCs as well as in RA synovial tissue cells. The addition of either TNF blockers or dexamethasone suppressed lipopolysaccharide-induced mPGES-1 and COX-2 expression in synovial fluid monocyte/macrophages in vitro and decreased the production of PGE(2). Intraarticular treatment with glucocorticoids significantly reduced both mPGES-1 and COX-2 expression in arthritic synovial tissue ex vivo. The number of COX-1-expressing cells in synovial tissue was also significantly decreased by glucocorticoid treatment. In contrast, neither mPGES-1 nor COX-2 expression in synovial tissue was significantly suppressed by anti-TNF therapy. CONCLUSION: These data are the first to demonstrate the effects of antirheumatic treatments on mPGES-1 expression in RA and suggest that the inhibition of PGE(2) biosynthesis, preferably by targeting mPGES-1, might complement anti-TNF treatment for optimal antiinflammatory results in RA.

Adult↗

PubMed1991

Effect of rat plasma high density lipoprotein with or without apolipoprotein E on the cholesterol uptake and on the induction of the corticosteroid biosynthetic pathway in newborn rat adrenocortical cell cultures.

High density lipoprotein (HDL) has been shown to induce the cellular accumulation of cholesterol esters and the biosynthetis of 21-hydroxysteroids (corticosteroids) newborn rat adrenocortical cells cultivated in serum-free medium. In order to identify the component(s) of HDL responsible for these effects, we investigated the ability of rat HDL subfractions and HDL with or without apolipoprotein E to deliver cholesterol to cells and to stimulate the steroid biosynthetic pathways in adrenal cultured cells. The total cholesterol uptake from HDL2 was greater than that observed with HDL rich in apolipoprotein E (HDL1 and HDLc). Furthermore, the increase of the ratio between 21-hydroxysteroids and reductive metabolites of progesterone was higher with HDL2 than with HDL1 or HDLc. The results of competitive studies between LDL and HDL subfractions indicate that adrenal cells take up cholesterol from HDL2 and LDL by separate mechanisms but that LDL and HDL containing apolipoprotein E share the same uptake processes. In experiments with various concentrations of HDLc or HDL without apolipoprotein E, the adrenal cells displayed a higher affinity for rat HDLc than for rat HDL without apolipoprotein E. However, HDL without apolipoprotein E produced a higher enhancement of the cholesterol cell content and was 3-fold more effective in stimulating 21-hydroxylated steroid production than rat HDLc. Although these findings suggest a participation of HDL with apolipoprotein E in the HDL interaction with rat adrenal cells, the predominant effect on these cells is devoluted to HDL containing mainly apolipoprotein A.

Adrenal Cortex↗

PubMed2003

Semisynthetic production of unnatural L-alpha-amino acids by metabolic engineering of the cysteine-biosynthetic pathway.

There is an increasing demand for peptide-mimicking molecules to modulate the interactions between proteins of pharmaceutical and agrochemical interest and their target polypeptides. Unnatural L-alpha-amino acids differing from the 20 naturally proteinogenic amino acids only in their side chain are ideal for this purpose, but their chemical synthesis is complex. Here we describe a fermentation-based approach for biosynthesis of unnatural amino acids after re-engineering the cysteine-biosynthetic pathway in Escherichia coli. O-acetylation of serine, the committed step of the pathway, was released from feedback inhibition by mutating the serine acetyltransferase gene. Next, the naturally broad substrate specificity of O-acetylserine sulfhydrylase was exploited for the direct in vivo incorporation of an unnatural side chain in a semisynthetic fermentation process comparable to the production of beta-lactams. O-acetyl-L-serine extruded from the cells by way of the O-acetylserine efflux protein was amenable to further biotransformations.

Amino Acids↗