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PubMed2003

[Expression and assessment of double genes of tyrosine hydroxylase gene and aromatic L-amino acid decarboxylase gene in vitro].

The characteristic pathological changes of Parkinson s disease (PD) include a severe loss of dopamine neurons in the substantia nigra and a severe decrease in dopamine in the striatum. Since the expression of tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) in the biosynthetic pathway for dopamine are low, a promising approach to the gene therapy of PD is to augment the gene expression of the enzymes in the biosynthetic pathway for dopamine. In the present study, human TH and AADC genes were reconstructed into retrovirous vectors pLHCX and pLNCX(2) respectively. Then pLHCX/TH and pLNCX(2)/AADC were transfected into packaging cell line PA317 with liposome. PA317/TH and PA317/AADC were selected by different antibiotics. Gene expression was examined by methods of immunohistochemistry and in situ hybridization. The catalytic activity of two cloned gene enzymes was assessed in vitro by HPLC-EC. Immunocytochemical staining showed that TH and AADC were expressed efficiently in vitro. Both TH and AADC mRNA were transcripted in PA317 cell lines by using in situ hybridazation. HPLC-EC experiments revealed that the transfected cells produced a significantly higher level of dopamine and L-dopa than the untransfected cells. The two genetically modified cells could improve the production of L-dopa and dopamine in response to suitable substrate. The present results suggest that not only recombinant TH and AADC genes are successfully expressed in vitro, but also the enzymes have respective functional activities. These results have set up a way for in vivo gene therapy of PD with TH and AADC genes.

Aromatic-L-Amino-Acid Decarboxylases↗

PubMed1994

Insertional activation of mevalonate kinase by hepatitis B virus DNA in a human hepatoma cell line.

Insertional mutagenesis of growth related genes by hepatitis B virus (HBV) DNA is presumed to play a role in hepatocarcinogenesis. Here, we report on insertional activation of the mevalonate kinase (MK) gene in the human hepatoma cell line PLC/PRF/5. Integration of HBV DNA dissociated the promoter and upstream regulatory elements of the gene from its coding sequences. This led to the over-expression of hybrid transcripts arising from an HBV promoter and the consequent over-production of functionally active mevalonate kinase. MK phosphorylates mevalonate, a major intermediate in the branched cholesterol/isoprenoid biosynthetic pathway. Isoprenylation is crucial to the functions of cellular proteins related to growth control, including the proto-oncogene ras. As the enzymes of these biosynthetic pathways are regulated at multiple points by negative feedback, both transcriptionally and at the protein level, the results discussed here support the idea that aberrant growth could result from deregulated overexpression of MK and, perhaps, other enzymes in the cholesterol pathway. These results invoke novel mechanisms by which cell transformation might occur.

Amino Acid Sequence↗

PubMed1998

Recurrent missense mutation in the protoporphyrinogen oxidase gene underlies variegate porphyria.

The porphyrias represent a heterogeneous group of disorders of porphyrin or porphyrin-precursor metabolism, resulting from the inherited or acquired dysregulation of one of the eight enzymes in the porphyrin-heme biosynthetic pathway. Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of the penultimate enzyme in the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO). Recently, VP has been linked to the PPO gene on chromosome 1q22-23, and several disease-causing mutations have been described. In this study, we identified the underlying genetic lesion in two unrelated patients with VP and investigated all available family members by polymerase chain reaction, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. Mutation analyses in both families revealed a G-to-A transition in exon 6 of the PPO gene resulting in the substitution of arginine by histidine at position 168 of the protein (R168H). This arginine residue is evolutionarily conserved in human, mouse, and Bacillus subtilis, indicating the importance of this residue in PPO function. Our study establishes a recurrent missense mutation as the underlying genetic defect in two unrelated patients with VP and explains the occurrence of the phenotype in their families.

Adult↗

PubMed1994

Directed mutational analysis of bacteriochlorophyll a biosynthesis in Rhodobacter capsulatus.

Previous studies have established that most if not all of the genes required for synthesis of the Rhodobacter capsulatus essential photosystem are clustered on a 46 kb region of the chromosome known as the photosynthesis gene cluster. This region has recently been sequenced in its entirety by Hearst and co-workers, revealing the existence of 23 open reading frames, many of which are thought to be involved in the synthesis of bacteriochlorophyll. In this study we have undertaken a systematic directed mutational analysis of 12 open reading frames in the photosynthesis gene cluster to evaluate whether individual open reading frames have a role in photopigment biosynthesis. The results of this analysis demonstrate that mutations constructed in seven open reading frames resulted in a loss of bacteriochlorophyll biosynthesis, concomitant with the accumulation of specific intermediates in the Mg-tetrapyrrole biosynthetic pathway. One mutation was observed to result in partial disruption of bacteriochlorophyll biosynthesis, leading to the accumulation of bacteriochlorophyll as well as an intermediate in the biosynthetic pathway. We also observed that disruptions constructed in four open reading frames had no discernible effect on the synthesis of photopigments. The results of this analysis are discussed with regard to our current understanding of the role of each of these open reading frames in the synthesis of the R. capsulatus photosystem.

Bacterial Proteins↗

PubMed2003

QTL and candidate genes phytoene synthase and zeta-carotene desaturase associated with the accumulation of carotenoids in maize.

Carotenoids are a class of fat-soluble antioxidant vitamin compounds present in maize ( Zea mays L.) that may provide health benefits to animals or humans. Four carotenoid compounds are predominant in maize grain: beta-carotene, beta-cryptoxanthin, zeaxanthin, and lutein. Although beta-carotene has the highest pro-vitamin A activity, it is present in a relatively low concentration in maize kernels. We set out to identify quantitative trait loci (QTL) affecting carotenoid accumulation in maize kernels. Two sets of segregating families were evaluated-a set of F2:3 lines derived from a cross of W64a x A632, and their testcross progeny with AE335. Molecular markers were evaluated on the F2:3 lines and a genetic linkage map created. High-performance liquid chromatography was performed to measure beta-carotene, beta-cryptoxanthin, zeaxanthin, and lutein on both sets of materials. Composite interval mapping identified chromosome regions with QTL for one or more individual carotenoids in the per se and testcross progenies. Notably QTL in the per se population map to regions with candidate genes, yellow 1 and viviparous 9, which may be responsible for quantitative variation in carotenoids. The yellow 1 gene maps to chromosome six and is associated with phytoene synthase, the enzyme catalyzing the first dedicated step in the carotenoid biosynthetic pathway. The viviparous 9 gene maps to chromosome seven and is associated with zeta-carotene desaturase, an enzyme catalyzing an early step in the carotenoid biosynthetic pathway. If the QTL identified in this study are confirmed, particularly those associated with candidates genes, they could be used in an efficient marker-assisted selection program to facilitate increasing levels of carotenoids in maize grain.

Alkyl and Aryl Transferases↗

PubMed2005

Photoactivated perylenequinone toxins in fungal pathogenesis of plants.

Several genera of plant pathogenic fungi produce photoactivated perylenequinone toxins involved in pathogenesis of their hosts. These toxins are photosensitizers, absorbing light energy and generating reactive oxygen species that damage the membranes of the host cells. Studies with toxin-deficient mutants and on the involvement of light in symptom development have documented the importance of these toxins in successful pathogenesis of plants. This review focuses on the well studied perylenequinone toxin, cercosporin, produced by species in the genus Cercospora. Significant progress has been made recently on the biosynthetic pathway of cercosporin, with the characterization of genes encoding a polyketide synthase and a major facilitator superfamily transporter, representing the first and last steps of the biosynthetic pathway, as well as important regulatory genes. In addition, the resistance of Cercospora fungi to cercosporin and to the singlet oxygen that it generates has led to the use of these fungi as models for understanding cellular resistance to photosensitizers and singlet oxygen. These studies have shown that resistance is complex, and have documented a role for transporters, transient reductive detoxification, and quenchers in cercosporin resistance.

Ascomycota↗

PubMed2002

The age-related discrepancy in the effect of neuropeptide Y on select catecholamine biosynthetic enzymes in the adrenal medulla and hypothalamus in rats.

The elevated levels of circulating catecholamines (CAs) with age may be related to the increased expression of CA biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta hydroxylase (DbetaH) in the adrenal medulla of senescent compared with younger animals. Neuropeptide Y (NPY) is co-synthesized and co-released with CAs in the adrenal medulla. NPY inhibits the stimulated secretion of CAs, however, its role in regulation of the genes encoding CA biosynthetic enzymes is not clear. We hypothesized that NPY up-regulates TH, DbetaH and NPY expression in the adrenal medullae of young and old Fischer-344 rats. NPY increased mRNA expression of TH, DbetaH, NPY and also enhanced TH protein level in the adrenal medullae of young rats by 50%, 35%, 45% and by 20%, respectively. We also examined the effect of NPY on TH and NPY mRNA in the hypothalamus. Basal expression of TH mRNA was decreased in the hypothalamus with age. DNA binding activities of activator protein-1 and cAMP response element binding protein were also augmented only in the young by 140% and 125%, respectively. We conclude that NPY stimulates the CA biosynthetic pathway in the adrenal medulla and positive auto-regulation of NPY might be involved in this process. The stimulatory effect of NPY on adrenomedullary CA biosynthetic pathway is blunted with age.

Adrenal Medulla↗

PubMed1995

Uptake of exogenous sn-1-acyl-2-lyso-phosphatidylinositol into HeLa S3 cells. Reacylation on the cell surface and metabolism to glucosaminyl(acyl)phosphatidylinositol.

A HeLa S3 subline is unusual in accumulating relatively large amounts of glucosaminyl(acyl)phosphatidylinositol (GlcN(acyl)PI), a derivative of phosphatidylinositol (PI) in which both GlcN and a fatty acid are linked to inositol hydroxyl groups (D. Sevlever, D. Humphrey, and T.L. Rosenberry, submitted for publication). This lipid is a proposed intermediate on the biosynthetic pathway for glycosyl-PI (GPI) anchors of membrane proteins. In this study we demonstrate for the first time that exogenous inositol phospholipids can enter this biosynthetic pathway and be metabolized to GlcN(acyl)PI. When HeLa S3 cells were incubated for 24 h with exogenous PI or sn-1-acyl-2-lyso-phosphatidyl-inositol (lyso-PI) labeled with 3H in the inositol group, 25-30% of the label was recovered in cell-associated lipids and most of the remaining 70-75% in hydrophilic metabolites in the medium. The predominant labeled lipid was PI, with smaller amounts of lyso-PI, phosphatidylinositol 4-phosphate (PIP), and GlcN(acyl)PI. Both exogenous lipid precursors gave the same distribution of labeled lipids, and a similar distribution was observed for endogenous inositol phospholipids metabolically labeled with [3H]inositol. Addition of excess inositol had no effect on the conversion of [3H]lyso-PI to [3H]GlcN(acyl)PI, indicating that the conversion did not result from breakdown to [3H]inositol followed by resynthesis. The cellular orientation of incorporated PI and lyso-PI was determined by incubating cells at 4 degrees C with PI-specific phospholipase C (PI-PLC). This enzyme cleaves only PI and lyso-PI on the outer leaflet of the cell membrane. After 24-h incubation with either precursor, only about 15% of cell-associated [3H]PI or [3H]lyso-PI was on the outer leaflet. However, more than 60% of the [3H]PI was on the outer leaflet after 1-h incubation with either precursor, suggesting that substantial sn-2 acylation of exogenous [3H]lyso-PI occurred in the outer leaflet. This suggestion was confirmed by examining labeled lipids in cells after uptake of [3H]lyso-PI at 4 degrees C. No transmembrane translocation of lyso-PI, PI phosphorylation, or PI glycosylation occurred at this temperature, but some sn-2 acylation was apparent and more than 90% of the [3H]PI formed was on the outer leaflet. These data indicate that sn-2 acylation can occur in the outer leaflet of the cell membrane, perhaps by transacylation from other cell surface phospholipids.

Acylation↗

PubMed1989

The regulation of the 17-deoxy steroids in man.

One must consider the 17-DOS as a biosynthetic pathway with multiple regulatory factors. ACTH is its dominant regulator but in the absence of ACTH and in conditions where plasma renin activity is increased, this biosynthetic pathway maintains its sensitivity to exogenous ACTH. Suppression of the renin system delays the general recovery of aldosterone after the removal of an aldosterone producing adenoma but not of the 17-DOS: a pattern also showed after the removal of a DOC-producing adenoma. In addition to the possible role of the renin system there remain inexplicable situations in its regulation that cannot be explained by ACTH and renin. Our studies suggest that a non-renin, non-ACTH factor may influence the basal production of these steroids, and by its reduction, permits deviation of steroid substrate to cortisol production. This sequence may be operative in the "stress syndrome". Finally, one of the more interesting phenomenonologic patients who has been observed is a young male who has the biochemical findings and clinical signs of DOC excess with hypertension, hyperplasia, suppression of aldosterone and the RAS, and normal cortisol levels. All the 17-DOS are elevated and both adrenal veins have high concentrations. He represents excessive stimulation of this pathway by putative 17-deoxy regulator excess. The renin system is suppressed and ACTH levels are normal. Treatment with suppressive doses of glucocorticoid hormones diminishes the elevated 17-DOS and cortisol and ameliorates blood pressure. In summary, there seems to occur in clinical disorders and contrived experimental settings, suggestions that a non-renin, non-ACTH factor can regulate the 17-DOS, absence can explain some of the unusual conditions described (Fig. 1). The 17-DOS, while a vestigial pathway, may still cause disease, and provide clues to central organization of the adreno-cortical response to injury, stress, and disease.

Acquired Immunodeficiency Syndrome↗

PubMed1996

Proline biosynthesis in Streptococcus thermophilus: characterization of the proBA operon and its products.

The presence of proline in the medium was not essential for growth of Streptococcus thermophilus, indicating that there is a proline biosynthetic pathway in this organism. Genetic and biochemical analysis identified and characterized this pathway. Two genes, designated proB and proA, were cloned, sequenced and characterized. Biochemical analysis of the proB- and proA-encoded enzymes showed that the proline biosynthetic pathway of S. thermophilus is similar to the one previously described in Escherichia coli. The deduced amino acid sequence of a 2-408 kb DNA region containing the genes revealed the similarity of the S. thermophilus gene products to ProB and ProA of E. coli and Serratia marcescens, and to the corresponding N- and C-terminal domains of the bifunctional plant enzyme delta 1-pyrroline-5-carboxylate synthetase of Vigna aconitifolia. Northern blot analysis showed that the two genes in S. thermophilus are organized in a single operon with proB proximal and proA distal to the promoter; primer extension analysis indicated that proBA transcription is not under repressive control by exogenously supplied proline.

Aldehyde Oxidoreductases↗

PubMed1987

Sterol biosynthesis via cycloartenol and other biochemical features related to photosynthetic phyla in the amoeba Naegleria lovaniensis and Naegleria gruberi.

The sterols and sterol precursors of two amoebae of the genus Naegleria, Naegleria lovaniensis and Naegleria gruberi were investigated. Cycloartenol, the sterol precursor in photosynthetic organisms, is present in both amoebae. In N. lovaniesis, it is accompanied by lanosterol and parkeol, as well as by the 24,25-dihydro derivatives of these triterpenes. One of the most striking features of these amoebae is the accumulation of 4 alpha-methylsterols which are present in similar amounts as those of 4,4-desmethylsterols (3-5 mg/g, dry weight). 4 alpha-Methylergosta-7,22-dienol was identified as a new compound. Ergosterol was the major 4,4-desmethylsterol, accompanied by small amounts of C27 and other C28 sterols. Treatment of N. lovaniensis with fenpropimorph modified the sterol pattern of this amoeba and inhibited its growth. This fungicide, known to inhibit steps of sterol biosynthesis in fungi and plants, induced the disappearance of 4 alpha-methyl-delta 7-sterols and the appearance of the unusual delta 6,8,22-ergostatrienol as in A. polyphaga. These results might be explained by a partial inhibition of the delta 8----delta 7 isomerase, the small amounts of delta 7-sterols formed being converted into ergosterol which is still present in fenpropimorph-exposed cells. De novo sterol biosynthesis in N. lovaniensis was shown by incorporation of [1-14C]acetate into sterols and sterol precursors, especially cycloartenol. Lanosterol and parkeol were not significantly labelled. Furthermore, [3-3H]squalene epoxide was efficiently cyclized by a cell-free system of this amoeba into cycloartenol, and again no significant radioactivity was detected in lanosterol and parkeol. This shows that cycloartenol, the sterol precursor in plants and algae, is also the sterol precursor in Naegleria species, and that these amoebae, like A. polyphaga, are related by some biosynthetic pathways to photosynthetic phyla. Lanosterol, the sterol precursor in non-photosynthetic phyla (animal and fungi) and parkeol are more likely dead-ends of this biosynthetic pathway. The peculiar phylogenetic position of these protozoa was further emphasized by the action of indole acetic acid and other auxine-like compounds on their growth. Indeed amoebic growth was enhanced in the presence of these higher plant growth hormones. The differences in the sterol composition of the protozoa we have hitherto examined is related to their sensitivity toward polyene macrolide antibiotics.(ABSTRACT TRUNCATED AT 400 WORDS)

Amoeba↗

PubMed1984

Cholesterol-induced growth stimulation, cell aggregation, and membrane properties of ascites tumor cells in culture.

Ascites tumor cells can be cultivated at a reduced serum concentration if cholesterol (2.50 mg per 100 ml of medium) is added to the culture medium. At serum concentrations of 3%, optimal growth properties are obtained; below 3%, cell cultures usually perish after a few days. Cells grown in the presence of added cholesterol have an elevated content of this molecule per cell as well as in the plasma membrane, and they also show a cholesterol concentration-dependent rate of proliferation. Precursors of the cholesterol-biosynthetic pathway like mevalonic acid, added in mM amounts, or squalene and lanosterol cannot be substituted for cholesterol itself. This is due to the observation that the biosynthetic pathway is blocked at the stage of lanosterol conversion to cholesterol. Cholesterol de novo synthesis from acetate is regulated by the cholesterol content of the cells, which also affects the production of ubiquinone and dolichol. Growth factors such as insulin, prostaglandin F2 alpha, and transferrin added to the medium do not mimic the cholesterol-induced effect. Distribution of DNA during cell cycle and the cell density-dependent reduction in macromolecule synthesis is very similar to the control cells. In contrast, cells without added cholesterol show reduced growth properties accompanied by the accumulation of cells in the mitotic and G2 phase. The cholesterol/phospholipid ratio of the plasma membranes of cholesterol-rich cells is about 15% lower than of the control cells and 40% higher compared to the cholesterol-poor cells, which, however, does not significantly alter the membrane fluidity between the cholesterol-rich and -poor cells as revealed by fluorescence polarization measurements. The most dramatic behavior of the cholesterol-rich cells is their tendency to form aggregates, which is demonstrated either by concanavalin A-induced agglutination or by cell density-dependent aggregation shown by interference microscopy in vivo.

Agglutination↗

PubMed2000

Clavulanic acid, a beta-lactamase inhibitor: biosynthesis and molecular genetics.

Clavulanic acid is a secondary metabolite produced by Streptomyces clavuligerus. It possesses a clavam structure and a characteristic 3R,5R stereochemistry essential for action as a beta-lactamase inhibitory molecule. It is produced from glyceraldehyde-3-phosphate and arginine in an eight step biosynthetic pathway. The pathway is carried out by unusual enzymes, such as (1) the enzyme condensing both precursors, N2-(2-carboxyethyl)-arginine (CEA) synthetase, (2) the beta-lactam synthetase cyclizing CEA and (3) the clavaminate synthetase, a well-characterized multifunctional enzyme. Genes for biosynthesis of clavulanic acid and other clavams have been cloned and characterized. They offer new possibilities for modification of the pathway and for obtaining new molecules with a clavam structure. The state of the regulatory proteins controlling clavulanic acid biosynthesis, as well as the relationship between the biosynthetic pathway of clavulanic acid and other clavams, is discussed.

Anti-Bacterial Agents↗

PubMed2005

Ten novel mutations in the molybdenum cofactor genes MOCS1 and MOCS2 and in vitro characterization of a MOCS2 mutation that abolishes the binding ability of molybdopterin synthase.

Molybdenum cofactor deficiency (MIM#252150) is a severe autosomal-recessive disorder with a devastating outcome. The cofactor is the product of a complex biosynthetic pathway involving four different genes (MOCS1, MOCS2, MOCS3 and GEPH). This disorder is caused almost exclusively by mutations in the MOCS1 or MOCS2 genes. Mutations affecting this biosynthetic pathway result in a lethal phenotype manifested by progressive neurological damage via the inactivation of the molybdenum cofactor-dependent enzyme, sulphite oxidase. Here we describe a total of ten novel disease-causing mutations in the MOCS1 and MOCS2 genes. Nine out of these ten mutations were classified as pathogenic in nature, since they create a stop codon, affect constitutive splice site positions, or change strictly conserved motifs. The tenth mutation abolishes the stop codon of the MOCS2B gene, thus elongating the corresponding protein. The mutation was expressed in vitro and was found to abolish the binding affinities of the large subunit of molybdopterin synthase (MOCS2B) for both precursor Z and the small subunit of molybdopterin synthase (MOCS2A).

Carbon-Carbon Lyases↗

PubMed2001

Active site analysis of the potential antimicrobial target aspartate semialdehyde dehydrogenase.

Aspartate-beta-semialdehyde dehydrogenase (ASADH) lies at the first branch point in the biosynthetic pathway through which bacteria, fungi, and the higher plants synthesize amino acids, including lysine and methionine and the cell wall component diaminopimelate from aspartate. Blocks in this biosynthetic pathway, which is absent in mammals, are lethal, and inhibitors of ASADH may therefore serve as useful antibacterial, fungicidal, or herbicidal agents. We have determined the structure of ASADH from Escherichia coli by crystallography in the presence of its coenzyme and a substrate analogue that acts as a covalent inhibitor. This structure is comparable to that of the covalent intermediate that forms during the reaction catalyzed by ASADH. The key catalytic residues are confirmed as cysteine 135, which is covalently linked to the intermediate during the reaction, and histidine 274, which acts as an acid/base catalyst. The substrate and coenzyme binding residues are also identified, and these active site residues are conserved throughout all of the ASADH sequences. Comparison of the previously determined apo-enzyme structure [Hadfield et al. J. Mol. Biol. (1999) 289, 991-1002] and the complex presented here reveals a conformational change that occurs on binding of NADP that creates a binding site for the amino acid substrate. These results provide a structural explanation for the preferred order of substrate binding that is observed kinetically.

Aspartate-Semialdehyde Dehydrogenase↗

PubMed2002

Structural studies of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: the apo, substrate, and product-aldimine complexes.

The evolution of biosynthetic pathways is difficult to reconstruct in hindsight; however, the structures of the enzymes that are involved may provide insight into their development. One enzyme in the cobalamin biosynthetic pathway that appears to have evolved from a protein with different function is L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica, which is structurally similar to histidinol phosphate aminotransferase [Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C., and Rayment, I. (2002) Biochemistry 41, 4798-4808]. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. To understand the relationship between this decarboxylase and the aspartate aminotransferase family to which it belongs, the structures of CobD in its apo state, the apo state complexed with the substrate, and its product external aldimine complex have been determined at 1.46, 1.8, and 1.8 A resolution, respectively. These structures show that the enzyme steers the breakdown of the external aldimine toward decarboxylation instead of amino transfer by positioning the carboxylate moiety of the substrate out of the plane of the pyridoxal ring and by placing the alpha-hydrogen out of reach of the catalytic base provided by the lysine that forms the internal aldimine. It would appear that CobD evolved from a primordial PLP-dependent aminotransferase, where the selection was based on similarities between the stereochemical properties of the substrates rather than preservation of the fate of the external aldimine. These structures provide a sequence signature for distinguishing between L-threonine-O-3-phosphate decarboxylase and histidinol phosphate aminotransferases, many of which appear to have been misannotated.

Binding Sites↗

PubMed2000

Mechanism of the incidental production of a melanin-like pigment during 6-demethylchlortetracycline production in Streptomyces aureofaciens.

The secondary metabolite 6-demethylchlortetracycline (6-DCT), which is produced by Streptomyces aureofaciens, is used as a precursor of semisynthetic tetracyclines. Strains that produce 6-DCT also produce a melanin-like pigment (MP). The correlation between MP production and 6-DCT production was investigated by using S. aureofaciens NRRL 3203. Production of both MP and 6-DCT was repressed by phosphate or ammonium ions, suggesting that syntheses of these compounds are controlled by the same regulators. Ten chlortetracycline-producing recombinants were derived from 6-DCT-producing mutant NRRL 3203 by gene replacement. All of the recombinants produced chlortetracycline but not MP, indicating that MP production is the results of a defect in the 6-methylation step and suggesting that the polyketide nonaketideamide is a common intermediate leading to MP as well as 6-DCT. To further examine the possibility that MP might be synthesized via the 6-DCT-biosynthetic pathway, mutants defective in 6-DCT biosynthesis were derived from a 6-DCT producer. Some of these mutants were able to produce MP, while others, including mutants with mutations in the gene encoding anhydrotetracycline oxygenase, an enzyme catalyzing the penultimate step in the pathway, produced neither 6-DCT nor MP. Production of 6-DCT and production of MP were restored simultaneously by integrative transformation with the corresponding 6-DCT-biosynthetic genes, indicating that some of 6-DCT-biosynthetic enzymes are indispensable for MP production. These findings suggest that a defect in the 6-methylation step results in redirection of carbon flux from a certain intermediate in the 6-DCT-biosynthetic pathway to a shunt pathway and results in MP production.

Anti-Bacterial Agents↗

PubMed1977

Chloroplast biogenesis. Net synthesis of protochlorophyllide from protoporphyrin IX by developing chloroplasts.

Developing chloroplasts were isolated from greening Cucumis cotyledons in a co-factor-enriched medium and were incubated in the dark with 14C-labeled and unlabeled protoporphyrin IX. The metabolic pools between protoporphyrin and protochlorophyllide were monitored spectrofluorometrically. The incorporation of the 14C label into protochlorophyllide was also determined. It was shown that protoporphyrin IX, a postulated intermediate of the chlorophyll biosynthetic pathway, was convertible into protochlorophyllide. Since protochlorophyllide is the immediate precursor of chlorophyll a it was concluded that protoporphyrin IX was indeed an intermediate of the chlorophyll biosynthetic pathway.

Chlorophyll↗