Isolation and Preparation of Pretyrosine, Accumulated as a Dead-End Metabolite by Neurospora crassa

Pretyrosine is an amino acid intermediate of phenylalanine and/or tyrosine biosyntheses in a variety of organisms. A procedure for the isolation of high-quality pretyrosine as the barium salt is described. Stable solutions of ammonium pretyrosine that are suitable for use as substrate in enzyme assays can be prepared in good yield with relatively few purification steps. A triple mutant of Neurospora crassa, bearing genetic blocks corresponding to each initial enzyme step of the three pathway branchlets leading to the aromatic amino acids, accumulates prephenate and pretyrosine. Although the time courses of prephenate and pretyrosine accumulations were found to be parallel in any given experiment, the ratios of the two metabolites varied as much as 100-fold depending upon such variables as carbon source, temperature of growth, accumulation, and especially the presence of aromatic pathway metabolites. Under appropriate nutritional conditions of accumulation, pretyrosine concentrations in excess of 4 mM in culture supernatant fluids were obtained. Strains individually auxotrophic for phenylalanine or tyrosine accumulate lesser amounts of prephenate and pretyrosine. The metabolic blocks of the mutant result in high intracellular levels of prephenate, which is then partially transaminated to pretyrosine. In N. crassa, pretyrosine is a dead-end metabolite since it is not enzymatically converted to phenylalanine or tyrosine. At a mildly acidic pH, pretyrosine is quantitatively converted to phenylalanine in a nonenzymatic reaction.     

Seasonal Variation in Serum Concentration of 5-S-Cysteinyldopa and 6-Hydroxy-5-Methoxyindole-2-Carboxylic Acid in Healthy Japanese

Serum concentrations of 5-S-cysteinyldopa (5-S-CD) and 6-hydroxy-5-methoxyindole-2-carboxylic acid (6H5MI2C) have been used as biochemical markers of melanoma progression. We examined the effect of solar radiation on serum levels of 5-S-CD and 6H5MI2C in 10 healthy Japanese by measuring these markers every month during a period of 2 years. 5-S-CD levels were higher in early summer and lower in early winter. The difference in the average levels was approximately twofold, but among the 240 samples, no individual values exceeded the upper limit of normal value, 10 nmol/L. A significant correlation (P<0.02) was observed between 5-S-CD level and solar radiation. 6H5MI2C levels showed a smaller variation than 5-S-CD. No correlation was observed between 6H5MI2C level and solar radiation. This study showed that serum 5-S-CD and 6H5MI2C in healthy Japanese did not exceed the upper limit of normal values even in sunny season.     

Isolation of o-Acetylbenzene-amidinocarboxylic Acid,a New Metabolite of Gibberella saubineti

The nickel requirement and the role of nickel were investigated in a recently identified oxygen-resistant hydrogen bacterium, Xanthobacter autotrophicus strain Y38. When 0.3 μm NiSO₄ was added to the basal medium which had not been supplemented with nickel, the cell concentration of autotrophically grown strain Y38 increased by about 4-fold and the resumption of cell growth occurred in the stationary phase. These results showed the requirement of nickel for the autotrophic growth of strain Y38. Since a trace of nickel was detected in the basal medium, the role of nickel was investigated using 0.2 mm or 0.4 mm EDTA-containing media. Other trace elements, Ca, Co, Cu, Mn, Mo and Zn, could not replace nickel. Nickel was not required for the heterotrophic growth of strain Y38. Nickel seems to be related a little to urease in strain Y38. Moderate hydrogenase induction was observed in hydrogenase deficient cells of strain Y38 under 95%H₂ + 5%O₂ when 300 μm NiSO₄ was added to 0.4 mm EDTA-containing buffer but it was completely inhibited by chloramphenicol, indicating that nickel was related to the hydrogenase synthesis. A nickel dependent increase in growth rate was demonstrated equally under 40%O₂ and 10%O₂, suggesting that nickel was not directly related to the oxygen-resistance of strain Y38.     

Formation of Thiazolidine-4-Carboxylic Acid Represents a Main Metabolic Pathway of 5-Hydroxytryptamine in Rat Brain

Incubation of 5-hydroxytryptamine (5-HT) with rat brain homogenate resulted in the formation of (4R)-2-[3'-(5'-hydroxyindolyl)-methyl]-1,3-thiazolidine-4-carboxyl ic acid (5'-HITCA) as the major metabolite. The substance represents the condensation product of 5-hydroxyindole-3-acetaldehyde with L-cysteine. The chemical structure was confirmed by chromatographic and chemical methods as well as by fast atom bombardment mass spectrometry. Incubation of 5-HT in the presence of L-cysteine yielded the thiazolidine as the main metabolite up to 4 h. Under these conditions, the concentration of 5-hydroxyindole-3-acetic acid (5-HIAA) amounted to about 20% and 57% of 5'-HITCA (0.5 h and 4 h, respectively). In contrast to these findings, indole-3-acetic acid (IAA) was identified as the major metabolite when tryptamine was incubated under similar conditions. (4R)-2-(3'-Indolylmethyl)-1,3-thiazolidine-4-carboxylic acid (ITCA) was found to be the main conversion product of tryptamine only during the first 30 min. To investigate the fate of the thiazolidines, radiolabelled and unlabelled ITCA was incubated with rat brain homogenate. The compound was degraded enzymatically and rapidly. Subcellular fractionation revealed that the enzyme activity was present mainly in the cytosolic fraction whereas the preparation of mitochondria showed less activity. The responsible enzyme is presumably a carbon-sulfur lyase (EC 4.4.1.-). The major metabolite was isolated by HPLC and identified by mass spectrometry as well as by comparison with reference compounds to be IAA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a Carboxylic Acid Metabolite from the Catabolism of Fluoranthene by a Mycobacterium sp

A Mycobacterium sp. previously isolated from oil-contaminated estuarine sediments was capable of extensively mineralizing the high-molecular-weight polycyclic aromatic hydrocarbon fluoranthene. A carboxylic acid metabolite accumulated and was isolated by thin-layer and high-pressure liquid chromatographic analyses of ethyl acetate extracts from acidified culture media. The metabolite reached a maximum concentration of approximately 0.65% after 24 h of incubation. On the basis of comparisons with authentic compound in which we used UV and fluorescence spectrophotometry and R_f values, as well as mass spectral and proton and carbon nuclear magnetic resonance spectral analyses, the metabolite was identified as 9-fluorenone-1-carboxylic acid. This is the first report in a microbial system of a fluoranthene metabolite in which significant degradation of one of the aromatic rings has occurred.     

Synthesis and Characterization of Melanins From Dihydroxyindole-2-Carboxylic Acid and Dihydroxyindole

Several studies have confirmed that a melanocyte-specific enzyme, dopachrome tautomerase (EC 5.3.2.3), catalyzes the isomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) (Pawelek, 1991). Here we report that DHICA, produced either enzymatically with dopachrome tautomerase or through chemical synthesis, spontaneously polymerized to form brown melanin that was soluble in aqueous solutions above pH 5. Under the same reaction conditions, solutions of either DOPA, DOPAchrome, or 5,6-dihydroxyindole (DHI) formed black, insoluble melanin precipitates. When DHICA and DHI were mixed together, with DHICA in molar excess, little or no precipitation of DHI-melanin occurred and the rate and extent of soluble melanin formation was markedly enhanced over that achieved with DHICA alone, suggesting co-polymerization of DHICA and DHI. With or without DHI, DHICA-melanins absorbed throughout the ultraviolet and visible spectra (200-600 nm). The DHICA-melanins precipitated below pH 5, at least in part because of protonation of the carboxyl groups. DHICA-melanins could be passed through 0.22 μm filters but could not be dialyzed through semi-permeable membranes with exclusion limits of 12,000-14,000 daltons. HPLC/molecular sieve analyses revealed apparent molecular weights ranging from 20,000 to 200,000 daltons, corresponding to 100-1,000 DHICA monomers per molecule of melanin. DHICA-melanins were stable to boiling, lyophilization, freezing and thawing, and incubation at room temperature for more than 1 year. The natural occurrence of oligomers of DHICA was first reported by Ito and Nichol (1974) in their studies of the brown tapetal pigment in the eye of the sea catfish (Arius felis L.). In experiments reported here, brown, but not black, melanins from mouse hairs, human melanoma cells, and peacock feathers were soluble in aqueous buffers. Since DHICA-melanins are both soluble and brown, the results raise the possibility that they are determinants of brown colors in the animal kingdom.     

Rhizonin, the First Mycotoxin Isolated from the Zygomycota, Is Not a Fungal Metabolite but Is Produced by Bacterial Endosymbionts

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this “first mycotoxin from lower fungi” is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.     

Some Effects of 1-Amino-2-Nitrocyclopentane-1-Carboxylic Acid on Flowering Plants

A study has been made of the effects on higher plants of i-amino-2-nitrocyclo-pentane-1-carboxylicacid (ANPCA), a metabolite of Aspergillus wentii Wehmer. ANPCAwas rapidly translocated to young tissues when injected intothe stems of pea seedlings, but when applied to the roots ofvery young seedlings only trace amounts entered the plants.In intact plants ANPCA produced loss of apical dominance andshorter internodes but was without effect on isolated stem orleaf sections. There was some reduction of cell extension inthe internodes of intact pea seedlings, but the main effectwas on cell numbers. ANPCA inhibited mitosis in root tips andapical buds, the degree of inhibition varying with the species.There appeared to be a block in the mitotic cycle between theend of DNA synthesis and prophase. Inhibition caused by ANPCAcould be reversed by L-leucine. Chlorophyll synthesis was inhibitedby ANPCA in newly formed tissue and in etiolated leaves transferredto the light. There was a considerable increase in the freeamino-acid pool and a decrease in gibberellin content due totreatment with ANPCA. It is suggested that the primary effectof ANPCA may be on protein or RNA synthesis.     

Fusamarin,a New Metabolite from a Species of Fusarium

Fusamarin, C₁₈H₂₄O₄, a metabolite of Fusarium sp., has been shown to be ((?)-(R)-(3)-trans-pentenyl-(3)-5-n-butyl-6,8-dihydroxy-3,4-dihydroisocoumarin) (1) on the basis of chemical and spectroscopic evidences.     

2-Amino-5-phosphono-3-pentenoic Acid,a New Amino Acid from N-1409 Substance,an Antagonist of Threonine

Discrimination of soy sauce samples consist of different 8 brands on the basis of principal components extracted from GC profiles of aroma concentrates was performed by stepwise discriminant analysis. Considering the results from sensory evaluation, classification of samples into 8, 3, and 2 groups was examined. Statistically significant difference was found among the 8 brands on the basis of principal components. The three groups consist of good, common, and inferior samples also classified correctly. Completely correct two way discrimination, good and bad, was accomplished in the same manner. These clear classifications suggested that evaluation and discrimination in sensory tests were performed by comparing the profiles of aroma substances.     

Application of Microbes and Microbial Esterases to the Preparation of Optically Active N-Acetylindoline-2-Carboxylic Acid

Methylotrophic bacteria, isolated from soil samples or from sewage sludge, proved to be useful sources of esterases for catalyzing the enantioselective hydrolysis of racemic N-acetyl-indoline-2-carboxylic acid methyl ester (7) to the corresponding (25) or (2R)-N- acetyl amino acid (6) with high optical yields. From the DMF-utilizer Pseudomonas DMF 5/8 and the methanol-utilizer Isolate EE 210, the corresponding esterases were isolated. Reactions with whole cells as well as with the purified enzymes are described.     

Characterization of Four Amino Acids Constituting Cyl-2, a Metabolite from Cylindrocladium scoparium

A biologically active metabolite designated Cyl-2 from a phytopathogenic fungus, Cylindrocladium scoparium, was found to be composed of four amino acid residues, which fully account for all of the atoms constituting Cyl-2. These amino acids were identified as L-pipecolic acid, L-isoleucine, D-O-methyltyrosine and 2-amino-8-oxo-9,10-epoxydecanoic acid. Thus, Cyl-2 was established as a cyclotetrapeptide containing novel amino acid residues.     

Isolation and Chemical Structure of New Oxidation Product of 5-Ketogluconic Acid,and a Hypothetical Pathway from Glucose to Tartaric Acid through this New Compound

In the course of study on the mechanism of the tartaric acid formation from 5-ketogluconic acid, a new intermediary substance with mauve color to Abdel-Akhel and Smith’s reagent was isolated from intact cell culture liquid. The chemical structure of this substance was determined as 1,2-dihydroxyethyl hydrogen L(+) tartrate from the results of hydrolysis experiments and from the identifications of the constituents of the molecule, and named “pretaric acid.” Tartaric acid was evidently produced from pretaric acid by intact cell culture. Clearly, then, pretaric acid appears to be an intermediate in the formation of tartaric acid from 5-ketogluconic acid. The authors assumed that in the formation of pretaric acid from 5-ketogluconic acid, a Baeyer-Villiger type oxidation occurred.     

Manganese Oxidation by Bacterial Isolates from the Indian Ridge System

The abundance and activity of culturable manganese-oxidizing bacteria were assessed from near-bottom water samples of the tectonically active Carlsberg Ridge. Retrievable counts as colony forming units (CFU) on dilute nutrient agar medium (dilNA = 2 gm l⁻¹ nutrient broth+2% agar) and on dilNA supplemented with 1, 2 and 3 mM MnCl₂·4H₂O were in the order of 10⁶ CFU l⁻¹. Retrievability of heterotrophs ranged from non-detectable levels (ND) to 2.82 × 10⁶ CFU l⁻¹. The retrievable counts on Mn amended dilNA ranged from ND to 3.21× 10⁶, 1.47 × 10⁶ and 1.45 × 10⁶ CFU l⁻¹ on 1, 2 and 3 mM, respectively. About 87% of the Mn tolerant isolates (n = 39) showed taxonomic affinities to Pseudomonas I and II sp. Two representative strains CR35 and CR48 (CR–Carlsberg Ridge) isolated on manganese-supplemented media were tested for their ability to tolerate a range of Mn amendments from 1 nM to 100 mM in terms of growth and respiration. CR35 represents 66% of the total CFU (3.04 × 10⁶ CFU l⁻¹), while CR48 represented only 6% of the total CFU (1.05 × 10⁶ CFU l⁻¹). The colonies of these two isolates were dark brown in color suggesting precipitation of Mn as oxide. Tests for the effect on growth and respiration were conducted in media simulating heterotrophic (amended with 0.01% glucose) and lithotrophic (unamended) conditions. Maximum stimulation in growth and respiration of CR35 occurred at 100 μM Mn both in unamended and amended media. At levels of Mn greater than 100 μM the counts decreased steadily. Total respiring cells of CR48 were stimulated to a maximum at 1 μM Mn in unamended medium and 1 nM in amended medium. Total cells counts for the same decreased beyond 100 μM Mn in unamended and 1 nM in amended medium. The isolates were tested for their ability to oxidize Mn ammendments from 1 μM to 10 mM Mn. At the end of a 76-day incubation period, there was evidence of manganese oxide precipitation at high Mn concentrations (≥1 mM) as a dark brown coloration on the sides of culture tubes. Highest Mn oxidation rates were observed at 10 mM Mn(II) concentration with CR35 oxidizing 27 and 25 μM Mn day⁻¹ in unamended and amended condition, respectively. CR48 oxidized Mn at the rate of 26 μM Mn day⁻¹ in unamended medium and 35 μM Mn day⁻¹ in amended medium. Scanning electron microscope (SEM) observations of both isolates revealed free-living cells in clustered matrices ≈2 μm diameter. Energy dispersive spectrum of the cell matrix of CR35 cultured in 1 mM Mn detected 30% Mn, while the cell aggregates of CR48 harbored 7–10% Mn. The relatively high specific activity of these mixotrophic bacteria under relatively oligotrophic conditions suggests that they may be responsible for scavenging dissolved Mn from the Carlsberg Ridge waters and could potentially participate in oxidation.     

Ornatipolide,a Novel Phenolic Metabolite from the Basidiomycete,Boletus ornatipes

A novel phenolic metabolite, ornatipolide, was isolated from the Boletus ornatipes fungus. Its structure was established by a combination of spectroscopic and chemical methods, and by an X-ray crystallographic analysis.     

Age-Dependent Discrimination between Stereoisomers of 1-Amino-2-Ethylcyclopropane-1-Carboxylic Acid in Carnation Petals

The ability of carnation petals (Dianthus caryophyllus L. cv White Sim) of different ages to convert the cis and trans isomers of 1-amino-2-ethylcyclopropane-1-carboxylic acid (AEC) to 1-butene was studied. Young petals, which produce ethylene at a low rate, convert both cis- and trans-AEC to 1-butene with low efficiency and at equal rates. In senescing petals, the rate of conversion of cis-AEC remains low, but there is a marked increase in the rate of trans-AEC conversion. Thus there is a clear evidence of stereodiscrimination between the isomers. Stimulating the rate of senescence by treatment with either 1-aminocyclopropane-1-carboxylic acid or ethylene further increases the rate of trans-AEC conversion. Delaying of petal senescence by silver thiosulphate or aminooxyacetic acid inhibits the rise in trans-AEC conversion.     

Identification of 3-Hydroxy-2-indolone-3-acetylaspartic Acid as a New Indole-3-acetic Acid Metabolite in Vicia Roots

A new indole-3-acetic acid (IAA) metabolite in the root of Viciafaba L. cv. Chukyo was identified as 3-hydroxy-2-indolone-3-acetylasparticacid, with the simpler name of dioxindole-3-acetylaspartic acid,by comparison with the authentic sample. Formation of dioxindole-3-aceticacid conjugates seems to be a major route of IAA metabolismin Vicia roots. (Received October 22, 1985; Accepted January 7, 1986)