Natural and directed biosynthesis of communesin alkaloids

A role for tryptophan, acetate, mevalonate and methionine in the biosynthesis of communesins A and B, novel structurally-related and biologically-active Penicillium metabolites, has been established by isotopic labelling techniques. The incorporation of (14)C-tryptamine has also been demonstrated. dl-2-(13)C-tryptophan specifically enriched two carbon atoms in the (13)C NMR spectrum, thereby defining the intra-molecular arrangement of the two tryptophan-derived moieties. Feeding differentially labelled precursors during communesin production showed that tryptophan and methionine are involved early in the biosynthesis and that mevalonate provides an isoprene which is added later. A biosynthetic pathway involving an early precursor based on tryptophan is proposed. Indole-N-((13)C-methyl) tryptophan was not incorporated into communesins implying that N-methylation of tryptophan is not the first step of the communesin biosynthetic pathway. During deamination of indole-N-((13)C-methyl) tryptophan to 1-(13)C-methylindole-3-carboxylic acid communesin biosynthesis was inhibited. Of several halogenated indoles tested for directed biosynthesis, only dl-6-fluoro-tryptophan and 6-fluoro-tryptamine caused accumulation of the corresponding monofluoro-analogues of communesins A and B.     

New insecticidal compounds, communesins C, D and E, from Penicillium expansum link MK-57

Three new communesin congeners, communesins C, D, and E, together with two known communesins A and B were isolated from okara that had been fermented with Penicillium expansum Link MK-57. All these communesins showed the insecticidal activity against the silkworms.     

Isolation of auxotrophs and analysis of regulation of tryptophan biosynthesis in Zymomonas mobilis

Tryptophan auxotrophs were isolated and used to analyze the regulation of tryptophan biosynthesis in Zymomonas mobilis. Twelve tryptophan auxotrophs were cassified as trp E, B or A based on accumulation of, or growth on, indole and anthranilic acid. Trp B mutants were found to accumulate indole when grown on limiting, but not on excess tryptophan, suggesting that tryptophan plays a role in regulating its biosynthesis. Tryptophan synthase and indoleglycerol phosphate synthase specific activities were measured in the wild-type strain and two trp mutants grown in limiting or excess tryptophan. Neither activity was repressed by exogenous tryptophan.Abbreviations CDRP O-(carboxyphenol amino)-1 deoxyribulose 5-phosphate - IGPS indoleglycerol phosphate synthase - TS tryptophan synthase Dedicated in memory of Dr. O. H. Smith     

Genetic organization of the biosynthetic gene cluster for the indolocarbazole K-252a in <Emphasis Type="Italic">Nonomuraea longicatena</Emphasis> JCM 11136

Indolocarbazole metabolite K-252a is a natural product that was previously reported as a potent protein kinase C inhibitor with in vitro and in vivo potency. From a biosynthetic viewpoint, this compound possesses structurally interesting features such as an unusual furanosyl sugar moiety, which are absent in the well-studied staurosporine and rebeccamycin. A cosmid library from genomic DNA of Nonomuraea longicatena JCM 11136 was constructed and screened for the presence of genes to be involved in the biosynthesis of indolocarbazole K-252a. Using as a probe an internal fragment of vioB, a Chromobacterium violaceum gene encoding a multifunctional enzyme that catalyzes tryptophan decarboxylation and condensation reaction in violacein biosynthesis, we isolated a DNA region that directed the biosynthesis of K-252a when introduced into the heterologous expression host Streptomyces albus. Sequence analysis of 45 kb revealed genes for indolocarbazole core formation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes. The cloned genes should help to elucidate the molecular basis for indolocarbazole biosynthesis and generate new indolocarbazole analogues by genetic engineering.     

Amino acid supplementation reveals differential regulation of aflatoxin biosynthesis in <Emphasis Type="Italic">Aspergillus flavus</Emphasis> NRRL 3357 and <Emphasis Type="Italic">Aspergillus parasiticus</Emphasis> SRRC 143

Aflatoxins are toxic and carcinogenic secondary metabolites produced by the fungi Aspergillus flavus and Aspergillus parasiticus. To better understand the molecular mechanisms that regulate aflatoxin production, the biosynthesis of the toxin in A. flavus and A. parasticus grown in yeast extract sucrose media supplemented with 50 mM tryptophan (Trp) were examined. Aspergillus flavus grown in the presence of 50 mM tryptophan was found to have significantly reduced aflatoxin B₁ and B₂ biosynthesis, while A. parasiticus cultures had significantly increased B₁ and G₁ biosynthesis. Microarray analysis of RNA extracted from fungi grown under these conditions revealed 77 genes that are expressed significantly different between A. flavus and A. parasiticus, including the aflatoxin biosynthetic genes aflD (nor-1), aflE (norA), and aflO (omtB). It is clear that the regulatory mechanisms of aflatoxin biosynthesis in response to Trp in A. flavus and A. parasiticus are different. These candidate genes may serve as regulatory factors of aflatoxin biosynthesis.     

Effect of Chronic Corn Consumption on Serotonin Content of Rat Brain

SEROTONIN, a putative neurotransmitter in the mammalian central nervous system, is synthesized in the brain by the 5-hydroxylation and decarboxylation of the essential amino-acid L-tryptophan^1,2. The control of serotonin biosynthesis seems to involve a different mechanism from that responsible for catecholamine biosynthesis^3,4 in its dependence on the availability of the amino-acid precursor^5,6. Thus, small doses of tryptophan that do not increase brain or plasma tryptophan concentrations beyond their normal daily ranges cause significant increases in the serotonin concentration of rat brain⁷. Conversely, the chronic ingestion of diets lacking in tryptophan (with casein hydrolysates or amino-acid mixtures substituted for natural proteins) depresses brain serotonin levels^8–10. The dependence of serotonin biosynthesis on tryptophan availability probably arises from the unusually high substrate K _M that characterizes tryptophan hydroxylase¹. It seems likely that this enzyme normally functions in an unsaturated state; hence physiological increases in intraneuronal tryptophan could drive the hydroxylation of the amino-acid and, ultimately, its conversion to serotonin.     

Tryptophan degradation in Saccharomyces cerevisiae: Characterization of two aromatic aminotransferases

Tryptophan was found to be degraded in Saccharomyces cerevisiae mainly to tryptophol. Upon chromatography on DEAE-cellulose two aminotransferases were identified: Aromatic aminotransferase I was constitutively synthesized and was active in vitro with tryptophan, phenylalanine or tyrosine as amino donors and pyruvate, phenylpyruvate or 2-oxoglutarate as amino acceptors. The enzyme was six times less active with and had a twenty times lower affinity for tryptophan (K _m=6 mM) than phenylalanine or tyrosine. It was postulated thus that aromatic aminotransferase I is involved in vivo in the last step of tyrosine and phenylalanine biosynthesis. Aromatic aminotransferase II was inducible with tryptophan but also with the other two aromatic amino acids either alone or in combinations. With tryptophan as amino donor the enzyme was most active with phenylpyruvate and not active with 2-oxoglutarate as amino acceptor; its affinity for tryptophan was similar as for the other aromatic amino acids (K _m=0.2–0.4 mM). Aromatic aminotransferase II was postulated to be involved in vivo mainly in the degradation of tryptophan, but may play also a role in the degradation of the other aromatic amino acids.A mutant strain defective in the aromatic aminotransferase II (aat2) was isolated and its influence on tryptophan accumulation and pool was studied. In combination with mutations trp2 ^fbr, aro7 and cdr1-1, mutation aat2 led to a threefold increase of the tryptophan pool as compared to a strain with an intact aromatic aminotransferase II.     

Control of Tryptophan Biosynthesis in Plant Tissue Cultures: Lack of Repression of Anthranilate and Tryptophan Synthetases by Tryptophan

Tobacco (cv. Xanthi and cv. Wisconsin 38), rice, carrot, tomato, and soybean tissue cultures were grown in liquid media containing L-tryptophan. The addition of tryptophan increased the cellular tryptophan levels greatly (12–2500 fold), but did not lower appreciably the levels of two tryptophan biosynthetic enzymes, anthranilate synthetase and tryptophan synthetase. However, the addition of 50 μM tryptophan to the crude enzyme extract completely inhibited the anthranilate synthetase activity while 1 mM tryptophan inhibited the tryptophan synthetase activity by only 10–20°/o. This information indicates that tryptophan biosynthesis is controlled by the feedback inhibition of anthranilate synthetase by tryptophan and not by repression of enzyme synthesis. All of the species had significant enzyme levels. Anthranilate synthetase activity could not be detected in extracts from cells grown on tryptophan unless the extracts were first passed through two G-25 Sephadex columns with a short 30 °C warming step in between, a procedure shown to remove an inhibitor of the enzyme.     

Polyamine Metabolism in Acanthamoeba: Polyamine Content and Synthesis of Ornithine,Putrescine, and Diaminopropane1

Five polyamines which could be separated by high performance liquid chromatography were found in Acanthamoeba castellanii (strain Neff). These included in order of decreasing abundance: 1,3-diaminopropane, spermidine, spermine, norspermidine, and putrescine. Only diaminopropane and norspermidine had been found previously. Spermine was present in cultures grown in broth, but not in defined medium. Radioactive substrates were used to establish that putrescine was synthesized by decarboxylation of ornithine, ornithine was synthesized from arginine or citrulline, and diaminopropane was synthesized from spermidine. The presence of ornithine decarboxylase (EC 4.1.1.17), arginase (EC 3.5.3.1), and urease (EC 3.5.1.5) and the absence of arginine decarboxylase (EC 4.1.1.19) were established. A scheme for polyamine biosynthesis in A. castellanii is proposed.     

Biosynthesis of Indoleacetic Acid from Tryptophan-C in Cell-free Extracts of Pea Shoot Tips

A 2-step, 1-dimensional thin-layer chromatographic procedure for isolating indoleacetic acid (IAA) was developed and utilized in investigations of the biosynthesis of IAA from tryptophan-¹⁴C in cell-free extracts of pea (Pisum sativum L.) shoot tips. Identification of a ¹⁴C-product as IAA was by (a) co-chromatography of authentic IAA and ¹⁴C-product on thin-layer chromatography, and (b) gas-liquid and thin-layer chromatography of authentic and presumptive IAA methyl esters. Dialysis of enzyme extracts and addition of α-ketoglutaric acid and pyridoxal phosphate to reaction mixtures resulted in approximately 2- to 3-fold increases in net yields of IAA over yields in non-dialyzed reaction mixtures which did not contain additives essential to a transaminase reaction of tryptophan. Addition of thiamine pyrophosphate to reaction mixtures further enhanced net biosynthesis of IAA. It is concluded that the formation of indolepyruvic acid and its subsequent decarboxylation probably are sequential reactions in the major pathway of IAA biosynthesis from tryptophan in cell-free extracts of Pisum shoot tips. Comparison of maximum net IAA biosynthesis in extracts of shoot tips of etiolated and light-grown dwarf and tall pea seedlings revealed an order, on a unit protein N basis, of: light-grown tall > light-grown dwarf > etiolated tall etiolated dwarf. It is concluded that the different rates of stem elongation among etiolated and light-grown dwarf and tall pea seedlings are correlated, in general, with differences in net IAA biosynthesis and sensitivity of the tissues to IAA.     

Fungicidal Activity of Two Spermidine Analogues

The potential use of polyamine analogues as inhibitors of polyamine biosynthesis to control plant pathogenic fungi is well established. However, all of this information relates to the use of putrescine analogues and no data exist for spermidine analogues. In the present work, two spermidine analogues. N¹- and N⁸-acetylspermidine were evaluated against powdery mildew on barley. Post-inoculation treatments reduced infection by 69.7% and 51.5%. respectively. Since the barley powdery mildew fungus cannot be grown axenically. mode of action studies were undertaken using the oat leaf-stripe pathogen Pyrenophora avenae. Neither of the analogues had any effect on polyamine biosynthesis in P. avenae grown in vitro. Although the mechanism(s) by which inhibitors affect in vivo fungal growth and in vitro growth may differ, it is unlikely that the antifungal properties of the analogues are the result of a perturbation in polyamine biosynthesis.     

<Emphasis Type="Italic">Withania coagulans</Emphasis> tryptophan decarboxylase gene cloning,heterologous expression,and catalytic characteristics of the recombinant enzyme

Tryptophan decarboxylase (EC 4.1.1.28) catalyzes pyridoxal 5′-phosphate (PLP)-dependent decarboxylation of tryptophan to produce tryptamine for recruitment in a myriad of biosynthetic pathways of metabolites possessing indolyl moiety. A recent report of certain indolyl metabolites in Withania species calls for a possible predominant functional role of tryptophan decarboxylase (TDC) in the genome of Withania species to facilitate production of the indolyl progenitor molecule, tryptamine. Therefore, with this metabolic prospection, we have identified and cloned a full-length cDNA sequence of TDC from aerial tissues of Withania coagulans. The functional WcTDC gene comprises of 1506 bp open reading frame (ORF) encoding a 502 amino acid protein with calculated molecular mass and pI value of 56.38 kDa and 8.35, respectively. The gene was expressed in Escherichia coli, and the recombinant enzyme was affinity-purified to homogeneity to discern its kinetics of catalysis. The enzyme (WcTDC) exhibited much higher K_m value for tryptophan than for pyridoxal 5′-phosphate and was dedicated to catalyze decarboxylation of only tryptophan or, to a limited extent, of its analogue (like 5-hydroxy tryptophan). The observed optimal catalytic functionality of the enzyme on the slightly basic side of the pH scale and at slightly higher temperatures reflected adaptability of the plant to hot and arid regions, the predominant natural habitat of the herb. This pertains to be the first report on cloning and characterization of heterologously expressed recombinant enzyme from W. coagulans and forms a starting point to further understanding of withanamide biosynthesis.     

Role of endogenous tryptophan during submerged fermentation of ergot alkaloids

Shake flask cultures ofClaviceps paspali (Stev. et Hall) andClaviceps purpurea (Fr.) Tul. on simple synthetic medium have been studied. Both strains grown in the absence of added tryptophan accumulate extra endogenous tryptophan. A certain concentration of cell-pool tryptophan is needed to promote alkaloid synthesis. Alkaloid production commences while tryptophan synthetase activity is increasing. In the alkloid-producing phase cell-pool tryptophan shows a single minimum while the change in level of cell-protein tryptophan is negligible. Alkaloid formation is suggested to reflect a regulatory device to keep endogenous tryptophan balanced. By adding amitrole the alkaloid spectrum is changed. The tryptophan-histidine cross-pathway probably serves a useful function inthe biosynthesis of ergot alkaloids.     

The regulation of tryptophan biosynthesis in Pseudomonas aeruginosa

Summary Eighteen auxotrophs of Pseudomonas aeruginosa requiring l-tryptophan for growth were isolated following nitrosoguanidine mutagenesis. Mutant blocks for each step of tryptophan biosynthesis were identified by enzymological assay. A regulatory mutant was characterized which was simultaneously constitutive for the gene products of trpA, trpB and trpD. Another class of regulatory mutant appears to synthesize tryptophan synthetase (i.e., trpE and trpF subunits) constitutively. The results implicate three control entities in the pathway of tryptophan biosynthesis: (i) The gene products of trpA, trpB and trpD are repressible by tryptophan, the range of enzyme specific activity varying at least fifty-fold. (ii) No regulation of the trpC gene product could be demonstrated, indicating that its synthesis is constitutive. (iii) The gene products of rpE and trpF are inducible by indoleglycerol 3-phosphate; the magnitude of induction can exceed 100-fold. These results together with some genetic data indicate a general similarity in gene-enzyme relationships between P. aeruginosa and P. putida. A number of specific differences that distinguish the two species are noted.A mutant blocked in the common pathway of aromatic biosynthesis was used to prove that enzymes of tryptophan biosynthesis other than tryptophan synthetase are not inducible by precursors of the common pathway such as chorismate. It is concluded that the concentration of tryptophan that signals total repression of the gene products of trpA, trpB and trpD is lower than the concentrations necessary for maximal feedback inhibition of anthranilate synthetase and for abolition of the induction of tryptophan synthetase.     

Physiological study of ergot: induction of alkaloid synthesis by tryptophan at the enzymatic level.

The enhancement of ergot alkaloid production by tryptophan and its analogues in both normal and high-phosphate cultures is more directly related to increased dimethylallyltryptophan (DMAT) synthetase activity rather than to a lack of regulation of the tryptophan biosynthetic enzymes. Thiotryptophan [beta-(1-benzo-thien-3-yl)-alanine] is rather ineffective in the end product regulation of tryptophan biosynthesis, whereas tryptophan and 5-methyltryptophan are potent effectors. The presence of increased levels of DMAT synthetase in ergot cultures supplemented with tryptophan or thiotryptophan, and to a lesser extent with 5-methyltryptophan, suggests that the induction effect involves de novo synthesis of the enzyme. Thiotryptophan and tryptophan but not 5-methyltryptophan can overcome the block of alkaloid synthesis by inorganic phosphate. The results with thiotryptophan indicate that the phosphate effect cannot be explained merely on the basis of a block of tryptophan synthesis.     

宽泛底物谱色氨酸脱羧酶体外合成卤代色胺衍生物和血清素

【背景】色氨酸脱羧酶在自然界有高度特异性,行使催化色氨酸为色胺的功能。一个色氨酸脱羧酶BaTDC参与南海海绵共生菌Bacillus atrophaeus C89次级代谢产物bacillamides的生物合成过程。【目的】探究BaTDC酶学特征和底物谱,建立体外合成色胺衍生物的方法。【方法】通过构建系统发育树揭示BaTDC在进化中的地位。在温度梯度和pH梯度下进行酶反应,利用不同的色氨酸衍生物为底物,通过HPLC和UPLC-MS检测酶反应过程,表征BaTDC活性。【结果】系统发育分析显示BaTDC与肠道菌Ruminococcus gnavus亲缘关系相近。纯化重组BaTDC的最适温度为40?45 °C,最适pH值为8.0。BaTDC可以催化羟代色氨酸和卤代色氨酸包括4-氟色氨酸和5,6,7-氯色氨酸及4-溴色氨酸,得到相应的卤代色胺衍生物和血清素。【结论】本研究分析了BaTDC的特性,发现BaTDC表现出宽泛的底物耐受性,可为前体喂养或定向生物合成新型药用色胺衍生物和下游复杂天然产物奠定基础。     

Synthesis of sugar-amino acid-nucleosides as potential glycosyltransferase inhibitors

Sugar-amino acid-nucleosides (SAAN) were synthesized to mimic glycosyl nucleotide donors based on the hypothesis that a basic amino acid may interact with carboxylate groups of the enzyme in a manner similar to the diphosphate metal ion complex. C-Glycoside analogues of the d-galactopyranose or l-arabinofuranose ring systems, and four amino acids (lysine, glutamine, tryptophan, and histidine), were chosen for this study. The targets were synthesized and tested against GlfT2, a galactofuranosyltransferase essential for cell wall galactan biosynthesis in Mycobacterium tuberculosis. The inhibition assay showed that analogues containing histidine and tryptophan are moderate inhibitors of GlfT2.     

Differential inhibition of indole-3-acetic acid and tryptophan biosynthesis by indole analogues. I. Tryptophan dependent IAA biosynthesis

Maize liquid endosperm extracts contain the enzymes necessary for all of the steps of the plant IAA biosynthetic pathway from tryptophan, and provide a means to assay the pathway in vitro. We have analyzed the reactions in the presence of a series of indole and indole-like analogues in order to evaluate the potential of these compounds to act as inhibitors of IAA biosynthesis. Such inhibitors will be useful to investigate the tryptophan to IAA pathway, to determine the precursors and intermediates involved, and to select for mutants in this process. A number of such compounds were tested using in vitro enzyme assays for both the tryptophan dependent IAA biosynthesis pathway and for tryptophan synthase activity. Some compounds showed strong inhibition of IAA biosynthesis while having only a slight effect on the reaction rate of tryptophan synthase . These results: (1) show that IAA biosynthesis can be selectively inhibited relative to tryptophan biosynthesis; (2) suggest potential ways to screen for IAA biosynthetic pathway mutations in plants; and (3) provide additional tools for studies of IAA biosynthesis in plants.     

Induction of serotonin biosynthesis is uncoupled from the coordinated induction of tryptophan biosynthesis in pepper fruits (<Emphasis Type="Italic">Capsicum annuum</Emphasis>) upon pathogen infection

It has been suggested that serotonin biosynthesis is regulated by tryptophan decarboxylase (TDC) in plants. To determine if TDC plays a rate-limiting role in serotonin biosynthesis, two TDC genes, PepTDC1 and PepTDC2, were cloned from pepper (Capsicum annuum L.) fruits infected with anthracnose fungus and their expression was then examined in various organs, including fruit that had been treated with the fungus or various chemicals. PepTDC1 expression was highly induced in pepper fruits after treatment with fungus and ethylene, while PepTDC2 was constitutively expressed at low levels in all pepper tissues. Additionally, predominant induction of PepTDC1 mRNA and TDC enzyme activity was detected in the unripe-green fruit, but not in the ripe-red fruit upon pathogen infection. Higher expression of TDC in unripe-green fruit was closely associated with increased levels of tryptamine, serotonin, and serotonin derivatives. However, unlike the enhanced serotonin synthesis, tryptophan levels responded unchanged when challenged with the pathogen in both the unripe-green fruit and the ripe-red fruit. Expression of two key tryptophan biosynthetic genes, anthranilate synthase (ASα) and tryptophan synthase (TSβ), remained unchanged in response to treatment. Also, anthranilate synthase enzyme activity remained steady regardless of pathogen infection. Taken together, these results suggest that the synthesis of serotonin was regulated by the induction of TDC without a simultaneous increase in tryptophan levels in pepper fruits.     

Biosynthesis of Indolocarbazole and Goadsporin,Two Different Heterocyclic Antibiotics Produced by Actinomycetes

The biosynthesis of staurosporine, rebeccamycin, and goadsporin, which are produced by actinomycetes and contain characteristic heterocyclic rings, was characterized by genetic methods. Staurosporine and rebeccamycin contain an indolocarbazole ring synthesized from two molecules of tryptophan, with indolepyruvic acid imine and chromopyrrolic acid as biosynthetic intermediates. A tetrameric hemoprotein synthesizes chromopyrrolic acid, and cytochrome P450 peroxidase catalyzes the intramolecular C–C coupling and decarboxylation of chromopyrrolic acid to form the indolocarbazole core. Goadsporin is a thiopeptide containing thiazole and oxazole heterocyclic rings. The structural gene godA is ribosomally translated to a goadsporin precursor peptide, and oxazole, methyloxazole, and thiazole rings are derived from serine, threonine, and cystein through post-translational modifications. On the basis of these knowledges, a wide variety of indolocarbazole and goadsporin analogs through the rational gene recombination and disruption of these biosynthetic genes were successfully produced.