Actinomycin synthesis in Streptomyces antibioticus. Purification and properties of a 3-hydroxyanthranilate 4-methyltransferase

A methyltransferase, which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate, has been purified to near homogeneity from 30-36-h mycelium of the bacterium Streptomyces antibioticus. The enzyme was obtained in approximately 20% yield with a purification of 130-fold. Polyacrylamide gel electrophoresis under denaturing conditions indicates that the enzyme is composed of a single subunit with Mr of about 36,000. On chromatography in 0.5 M NaCl, the enzyme displays a molecular weight of about 37,000. The specific activity of the enzyme in S. antibioticus mycelium is maximal between 30 and 36 h following inoculation of galactose/glutamic acid medium and, at those times post-inoculation, the specific activity is essentially the same in extracts of mycelium obtained from cultures grown on glucose rather than galactose as the carbon source. The enzyme activity is stimulated by Na2EDTA (in crude extracts) and by 2-mercaptoethanol and the methyltransferase shows a strong preference for HAA as substrate as compared with a number of HAA analogs. Thin layer chromatography of ethyl acetate extracts of large-scale incubation mixtures confirms that the product of the reaction is 4-methyl-3-hydroxyanthranilic acid. The reaction product was also a substrate for phenoxazinone synthase and was incorporated into actinomycin by S. antibioticus mycelium. Kinetic parameters for the methyltransferase reaction was determined.     

Actinomycin synthesis in Streptomyces antibioticus: enzymatic conversion of 3-hydroxyanthranilic acid to 4-methyl-3-hydroxyanthranilic acid.

A methyltransferase which utilizes 3-hydroxyanthranilic acid (HAA) as a substrate was identified in detergent-treated extracts of the bacterium Streptomyces antibioticus. The enzyme catalyzes the transfer of methyl groups from [14C]S-adenosylmethionine to HAA, but does not catalyze the methylation of 3-hydroxy-DL-kynurenine. Enzyme, substrate, time, and pH dependencies for the methyl transfer reaction were examined. Reaction products obtained from scaled-up reaction mixtures were fractionated by chromatography on Dowex 1, and the Dowex 1 fractions were examined by paper and thin-layer chromatography. One Dowex fraction was shown to contain a radioactive product with the chromatographic properties of 4-methyl-3-hydroxyanthranilic acid (MHA), a known intermediate in the biosynthesis of actinomycin. Available evidence indicates that the conversion of HAA to MHA is an early step in the biosynthesis of actinomycin by S. antibioticus and other actinomycin-producing streptomycetes.     

Production of Acidic Actinomycin Congeners by a Mutant Strain of Streptomyces antibioticus,No. B-1625

Streptomyces sp. No. B-1625, which was identified as a strain of Streptomyces antibioticus, is a typical producer of actinomycin, but also produces minor acidic antibiotic components (FA), besides actinomycins X₂, D and X_0β. The FA-components, which were obtained with a high-producing mutant, 11M-21, showed antibacterial and antitumor activities, and also similar visible and UV absorption spectra to those characteristic of actinomycin. The FA-components were separated into five components, FA₁ FA_2α, FA_2β, FA_3α and FA_3β, on TLC. Among them, one component, FA_3β, isolated in a purified state as an orange powder, has a composition of C, 52.97: H, 6.34: N, 10.48%, and is active against B. subtilis at a MIC of 5mcg/ml. The FA_3β component showed pK_a′ of 5.4 and 12.0 and λ_max at 443, 427 and 233 nm. From these properties, FA_3β is considered to be an acidic actinomycin congener.     

Biosynthesis of oleandomycin by cultures of Streptomyces antibioticus obtained after regeneration and fusion of protoplasts

The ability of Streptomyces antibioticus strains to synthesize oleandomycin is studied under the effect of regeneration and fusion of protoplasts. The production of strains-regenerants with an increased (by 30-50%) synthesis of oleandomycin is possible. Regenerants of mutants resistant to the proper antibiotic retain a high level of the oleandomycin synthesis more stably. Variations in the antibiotic-production ability are considered in regenerant populations of various generations.     

Incorporation of fluorotryptophan into triostin antibiotics by Streptomyces triostinicus

The quinoxaline chromophores of the antibiotics produced by Streptomyces triostinicus are derived from tryptophan. Protoplasts of this organism made novel products when they were incubated with DL-5-fluorotryptophan or DL-6-fluorotryptophan. When added to batch cultures of the organism, DL-5-fluorotryptophan, at concentrations as low as 10 microM, inhibited both mycelial growth and triostin production, but gave rise to novel products. These have been characterized, using fast atom bombardment mass spectrometry, as novel triostins in which one or both of the quinoxaline rings contain an atom of fluorine. The chromatographic properties of the triostins arising from the incorporation of DL-5-fluorotryptophan are very similar to those of triostins containing chlorine or bromine at position 6 of the quinoxaline ring; they are clearly different from those having a chlorine atom at position 7. Accordingly, it is suggested that the carbon atom at position 5 of the indole ring of tryptophan ends up at position 6 of the quinoxaline ring system in triostins A and C.     

Glutamate-induced uptake of proline by Streptomyces antibioticus.

Streptomyces antibioticus possesses an energy-dependent, carrier mediated transport system for the uptake of L-glutamate and L-proline. Amino acid transport was found to have a temperature optimum of 35 degrees C and a pH optimum from 7.0 to 8.0 for glutamate and 6.5 to 7.5 for proline uptake. Uptake did not depend upon Mg2+, Ca2+, Zn2+, Na+, or Fe2+ ions. Reversible p-hydroxymercuribenzoate inhibition of uptake indicated the involvement of an active sulfhydryl group. L-Glutamate uptake was mediated by a glutamate-inducible, nonspecific transport system, which was extremely stable and was not subject to substrate inhibition by L-proline. On the other hand, L-proline transport was mediated by at least two systems. The L-glutamate-inducible nonspecific system can account for uptake of proline by the mycelium grown in glutamate. In addition, a proline-specific, constitutive transport system was found to be present in the mycelium grown in organic and inorganic nitrogen sources other than L-glutamate. Shift experiments revealed that proline transport is not as stable as glutamate transport when the glutamate-inducible nonspecific system is utilized.     

Norepinephrine Metabolism in Man Using Deuterium Labelling: The Conversion of 4-Hydroxy-3-Methoxyphenylglycol to 4-Hydroxy-3-Methoxymandelic Acid

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with three deuterium atoms was used to study the disposition of peripherally administered HMPG. Five healthy men were given an intravenous pulse dose of 4.3 μmol of labelled HMPG and subsequent plasma and urine levels of endogenous and labelled HMPG as well as those of 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) were determined by gas chromatography-mass spectrometry, using selected ion detection. Approximately 40% of the injected amount of deuterium-labelled HMPG was recovered in the urine as HMMA and another 40% was eliminated as HMPG conjugates. Thus, the HMPG formed from norepinephrine either in the central or peripheral nervous system undergoes both conjugation and extensive oxidation.     

Biosynthesis of Polyketide Antibiotics by Various StreptomycesSpecies that Produce Actinomycins

A collection of actinomycin-producing Streptomycesstrains, their variants with different levels of antibiotic biosynthesis, and recombinant strains were screened in order to select new strains that produce polyketide antibiotics. Screening with the use of the cloned actgene encoding a component of actinorhodin polyketide synthase (PKS) multienzyme complex from Streptomyces coelicolorrevealed that many strains tested can synthesize polyketide antibiotics along with actinomycins. A relationship between the biosynthetic pathways of actinomycins and polyketides is discussed.     

Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus.

Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration.     

Genome-Based Analysis and Gene Dosage Studies Provide New Insight into 3-Hydroxy-4-Methylvalerate Biosynthesis in Ralstonia eutropha

Recombinant Ralstonia eutropha strain PHB⁻4 expressing the broad-substrate-specificity polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. strain 61-3 (PhaC1_Ps) synthesizes a PHA copolymer containing the branched side-chain unit 3-hydroxy-4-methylvalerate (3H4MV), which has a carbon backbone identical to that of leucine. Mutant strain 1F2 was derived from R. eutropha strain PHB⁻4 by chemical mutagenesis and shows higher levels of 3H4MV production than does the parent strain. In this study, to understand the mechanisms underlying the enhanced production of 3H4MV, whole-genome sequencing of strain 1F2 was performed, and the draft genome sequence was compared to that of parent strain PHB⁻4. This analysis uncovered four point mutations in the 1F2 genome. One point mutation was found in the ilvH gene at amino acid position 36 (A36T) of IlvH. ilvH encodes a subunit protein that regulates acetohydroxy acid synthase III (AHAS III). AHAS catalyzes the conversion of pyruvate to 2-acetolactate, which is the first reaction in the biosynthesis of branched amino acids such as leucine and valine. Thus, the A36T IlvH mutation may show AHAS tolerance to feedback inhibition by branched amino acids, thereby increasing carbon flux toward branched amino acid and 3H4MV biosynthesis. Furthermore, a gene dosage study and an isotope tracer study were conducted to investigate the 3H4MV biosynthesis pathway. Based on the observations in these studies, we propose a 3H4MV biosynthesis pathway in R. eutropha that involves a condensation reaction between isobutyryl coenzyme A (isobutyryl-CoA) and acetyl-CoA to form the 3H4MV carbon backbone.     

Formation of 3-Keto-4-deoxypentosone and 3-Hydroxy-2-pyrone by the Degradation of Dehydro-l-ascorbic Acid

A crystalline phenylhydrazone was obtained when a heated solution of dehydro-l-ascorbic acid (DHA) was treated with phenylhydrazine-HCl. Its molecular formula was C₁₇H₁₈N₄O₂, and the structure was determined to be 1,2-bis(phenylhydrazone) of 3-keto-4-deoxypentosone, a new tricarbonyl compound which was considered to be one of the possible intermediates of the browning reaction of DHA. 3-Hydroxy-2-pyrone was also isolated from the ether extract of the heated DHA solution as a main aroma compound produced from DHA. Possible formation mechanisms of these compounds were discussed.     

Biosynthesis of dioscorine : Incorporation of nicotinic acid into the isoquinuclidine moiety

The administration of nicotinic-[2-¹⁴C] acid to Dioscorea hispida plants afforded radioactive dioscorine (1.9% absolute incorporation) and a systematic degradation of the alkaloid indicated that essentially all the activity was located at C-3. Dioscorine derived from nicotinic-[5,6-¹⁴C, ¹³C₂] acid was also labelled. Its proton noise decoupled ¹³C NMR spectrum contained satellites at C-1 and C-7 due to spin-spin coupling of contiguous ¹³C atoms arising from direct incorporation of the labelled nicotinic acid. A biosynthetic scheme representing a novel utilization of nicotinic acid is proposed.     

CO2 Incorporation and 4-Hydroxy-2-Methylbenzoic Acid Formation during Anaerobic Metabolism of m-Cresol by a Methanogenic Consortium

The metabolism of m-cresol by methanogenic cultures enriched from domestic sewage sludge was investigated. In the initial studies, bromoethanesulfonic acid was used to inhibit methane production. This led to the accumulation of 4.0 ± 0.8 mol of acetate per mol of m-cresol metabolized. These results suggested that CO₂ incorporation occurred because each molecule of m-cresol contained seven carbon atoms, whereas four molecules of acetate product contained a total of eight carbon atoms. To verify this, [¹⁴C]bicarbonate was added to bromoethanesulfonic acid-inhibited cultures, and those cultures yielded [¹⁴C]acetate. Of the label recovered as acetate, 89% was found in the carboxyl position. Similar cultures fed [methyl-¹⁴C]m-cresol yielded methyl-labeled acetate. A ¹⁴C-labeled transient intermediate was detected in cultures given either m-cresol and [¹⁴C]bicarbonate or bicarbonate and [methyl-¹⁴C]m-cresol. The intermediate was identified as 4-hydroxy-2-methylbenzoic acid. In addition, another metabolite was detected and identified as 2-methylbenzoic acid. This compound appeared to be produced only sporadically, and it accumulated in the medium, suggesting that the dehydroxylation of 4-hydroxy-2-methylbenzoic acid led to an apparent dead-end product.     

Formation of 5-Hydroxy-4-Ketohexanoic Acid by Yeast

When grown on medium containing ethanol as the sole carbon source, three of five strains of yeast tested produced a keto acid which was demonstrated by paper chromatography. This compound was isolated and its structure was examined by elementary analysis, infrared spectrum, nuclear magnetic resonance and periodate oxidation. The compound was proved to be identical with 5-hydroxy-4-ketohexanoic acid. Formation of this compound by cell suspensions of Hansenula miso IFO 0146 was achieved by addition of acetaldehyde, although the presence of α-ketoglutaric acid enhanced the formation of the keto acid from acetaldehyde during a short incubation period. Added 5-hydroxy-4-ketohexanoic acid was exhausted by the cell suspension.     

Mechanism of 3-Methylanthranilic Acid Derepression of the Tryptophan Operon in Escherichia coli

3-Methylanthranilic acid (3MA) inhibits growth and causes derepression of the tryptophan biosynthetic enzymes in wild-type strains of Escherichia coli. Previous reports attributed this effect to an inhibition of the conversion of 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate to indole-3-glycerol phosphate and a consequent reduction in the concentration of endogenous tryptophan. Our studies have shown that 3MA-resistant mutants linked to the tryptophan operon have a feedback-resistant anthranilate synthetase; mutants with an altered indole-3-glycerol phosphate synthetase were not found. 3MA or 7-methylindole can be metabolized to 7-methyltryptophan, and 3MA, 7-methylindole, and 7-methyltryptophan lead to derepression of the tryptophan operon. Furthermore, 3MA-resistant mutants are also resistant to 7-methylindole derepression. These results strongly suggest that the primary cause of derepression by 3MA is through its conversion to 7-methyltryptophan, which can inhibit anthranilate synthetase, thereby decreasing the concentration of endogenous tryptophan. Unlike 5- or 6-methyltryptophan, 7-methyltryptophan does not appear to function as an active corepressor.