Edward Katz

Development of a chemically defined medium for the synthesis of actinomycin D by Streptomyces parvulus.

A chemically defined medium, consisting of d-fructose, l-glutamic acid, l-histidine, K2HPO4, MgSO4·7H2O, ZnSO4·7H2O, CaCl2·2H2O, FeSO4·7H2O, CoCl2·6H2O, and deionized water, was developed for synthesis of high yields (500 to 600 μg/ml) of actinomycin D by Streptomyces parvulus. Under these nutritional conditions, growth and actinomycin formation did not follow a typical trophophase-idiophase pattern. The amino acids appeared to have a sparing action on the utilization of d-fructose which was slowly and incompletely metabolized during mycelium development and antibiotic production. A significant repression of actinomycin synthesis by S. parvulus was observed when d-glucose (0.01 to 0.25%) was added to the culture medium. The repression was not due to a decline in the pH of the medium during glucose catabolism.

Changes in phenoxazinone synthetase activity during the growth cycle of Streptomyces antibioticus

Abstract Appearance of the enzyme phenoxazinone synthetase, which is involved in actinomycin formation, has been examined in cultures of Streptomyces antibioticus. De novo synthesis of this enzyme occurs between 9 and 36 hours after inoculation. Before this time the synthesis of the enzyme appears to be under “catabolite repression.” It has also been observed that at the time de novo synthesis of the enzyme begins, the organism becomes more resistant to actinomycin.

Inhibitory effect of actinomycin upon the producing organism

Abstract Actively growing cells of Streptomyces antibioticus are sensitive to actinomycin; by contrast, the organism appears to be quite insensitive to the compound during antibiotic production. Inhibition of actinomycin synthesis by 3-methyl- dl -proline is accompanied by an increase in cell growth. It is suggested that the antibiotic may function as a normal repressor substance in cellular metabolism.

Actinomycin Biosynthesis by Protoplasts Derived from Streptomyces parvulus

Conditions are described for the formation of protoplasts from Streptomyces parvulus that are able to synthesize actinomycin D de novo. Antibiotic synthesis by protoplasts, in contrast to that by mycelium, was sensitive to inhibition by actinomycin D and to a decrease in sucrose concentration. On the other hand, synthesis by mycelium was much more sensitive to inhibition by amino acid analogs (d-valine, cis-3-methylproline, and α-methyl-dl-tryptophan). In addition, the uptake of amino acids (l-methionine, sarcosine, and l- and d-valine) by protoplasts was significantly lower than that by mycelium. The advantages and limitations of using protoplasts for studying in vivo actinomycin synthesis are discussed. Images

Biosynthesis of polypeptide antibiotics

Peptide antibiotics possess a variety of chemical structures, e.g. cyclic diand oligopeptides. depsipeptides, linear peptides with repeating sequences of Land D-amino acids, substituted peptides containing non-peptide components. These antibiotics frequently contain amino acids which are not constituents of proteins. In general, biosynthesis of peptide antibiotics follows active growth and macromolecular synthesis by microorganisms. Studies to date indicate that the mechanism of formation of peptide antibiotics differs markedly from that of protein synthesis. In vivo experiments reveal that antibiotic synthesis is insensitive to inhibitors of protein and nucleic acid synthesis. Certain amino acid analogues can block either antibiotic or protein synthesis with minimal inhibition of the other process. Protein and peptide antibiotic synthesis probably compete for the intramolecular amino acid pool; when one process is inhibited there may be marked stimulation of the alternate system. In vitro experiments reveal that antibiotic synthesis is RNAse insensitive and there is no requirement for ribosomes, tRNA, or mRNA. Enzymatic synthesis generally requires ATP, Mg2 , a reducing agent, and the requisite amino acids. The process (gramicidin 5, tyrocidine) is catalysed by multi-enzyme complexes. The activation of amino acids is mediated by aminoacyl synthetases (in the protein complex) distinct from the aminoacyl-tRNA synthetases that activate amino acids for protein synthesis. The activated amino acids are transferred to thiol groups in the complex, and peptide synthesis occurs subsequently. The sequence of amino acids in the antibiotic is determined by the unique arrangement of specific enzymes in the multi-enzyme complex. Studies with cell-free systems as well as with intact organisms have revealed that chemically-related amino acids may substitute for normal constituents in antibiotic peptides. These data support the view that antibiotic peptide synthesis is catalysed by enzymes with relatively broad or low specificities. D-Amino acid biogenesis appears to involve an ATP-dependent racemization catalysed by a component of the multi-enzyme complex. Since the discovery of penicillin a large number of peptide antibiotics have been isolated and described in the literature16. These compounds, elaborated by a variety of microorganisms, contain one or more amino acids or moieties derived from amino acids. Studies have been carried out on the chemistry, biological activity, mode of action, and biosynthesis of these compounds. The evidence to date concerning the mechanism of formation of

Analysis of tyrosinase synthesis in Streptomyces antibioticus

Summary: 3 5S-labelling experiments and Western blot analysis were used to investigate methionine induction of tyrosinase synthesis in Streptomyces antibioticus. De novo synthesis of the enzyme occurred as a function of time and methionine concentration. Induction appeared to be relatively specific for methionine and closely related analogues. Under the conditions used, the enzyme was secreted rapidly, with little intracellular accumulation. Upon induction in the absence of Cu2+, apotyrosinase was synthesized at 70% of the level in control cultures provided with the cation. Inhibitor studies showed that both transcriptional and translational events are required for tyrosinase induction. Deletions in the ORF 438 region of the mel operon suggest that this sequence has a role in the phenotypic expression of tyrosinase.

Novel Actinomycins Formed by Biosynthetic Incorporation of cis- and trans-4-Methylproline

Streptomyces parvulus (Streptomyces parvullus) normally produces actinomycin D; in the presence of cis-4-methylproline, this species synthesizes two additional actinomycins, designated K1c and K2c, in which one and two proline sites, respectively, are occupied by cis-4-methylproline. Analogously, actinomycins K1t and K2t are formed in the presence of trans-4-methylproline. Both mixtures were separated chromatographically, and the four novel actinomycins were obtained in crystalline form. Their biological activities were compared with that of actinomycin D in respect to inhibition of ribonucleic acid, deoxyribonucleic acid, and protein synthesis and antimicrobial potency. In all cases examined, the order of activity D > K1t > K1c > K2t > K2c was observed, and the same sequence prevailed in a spectroscopic measure of their binding to deoxyribonucleic acid. In addition, proton nuclear magnetic resonance studies revealed that the replacement of proline by cis-4-methylproline alters the conformation of the antibiotic molecule.

Purification and characterization of tryptophan dioxygenase from Streptomyces parvulus

Tryptophan dioxygenase, derived from Streptomyces parvulus, was purified to near homogeneity and shown to have a native Mr of 88,000. Kinetic parameters of the enzyme were determined and evidence suggesting that it is a hemoprotein was obtained. Tryptophan dioxygenase has a high specificity toward L-tryptophan with an apparent Km of 0.3 mM. L-3-Hydroxykynurenine was a competitive inhibitor with respect to L-tryptophan with a Ki of 0.16 mM. In vitro, the enzyme displayed little activity in the absence of a reducing agent; ascorbate, at 50 mM, was the preferred reductant providing almost a 50-fold increase in enzyme activity. The regulation of tryptophan dioxygenase synthesis and activity is described. The expression of the enzyme is correlated with the biosynthesis of actinomycin D in S. parvulus. These results support the hypothesis that tryptophan dioxygenase functions as the first enzyme in the sequence converting L-tryptophan to the chromophore of this antibiotic.

Evidence for a constitutive and inducible form of kynurenine formamidase in an actinomycin-producing strain of Streptomyces parvulus.

Abstract Two forms of kynurenine formamidase have been found in an actinomycin-producing strain of Streptomyces parvulus . Formamidase I has a molecular weight of 42,000 and is synthesized constitutively. Formamidase II is smaller (24,000) and is present just prior to and during synthesis of actinomycin.

Biosynthesis of antibiotic peptides with isoleucine stereoisomers

Abstract Streptomyces antibioticus normally synthesizes an actinomycin mixture which contains N -methyl- l -valine and d -valine in the antibiotic peptides. Amino acid analyses indicate that trace amounts of isoleucine and, possibly, N -methylalloisoleucine are present also in actinomycin molecules. S. chrysomallus produces a number of actinomycins which contain either d -valine, d -alloisoleucine, or both amino acids in the molecule. During antibiotic synthesis in the presence of each of the four isomers of isoleucine, both organisms formed actinomycin mixtures which contained appreciable amounts of d -isoleucine and N -methyl- l -alloisoleucine. The conclusion that isoleucine possesses the d -configuration was based on studies of (1) optical rotary dispersion, (2) ion-exchange chromatography of appropriately synthesized dipeptides, and (3) reaction of the amino acid with d - and l -amino acid oxidases. Depending on the actinomycin-synthesizing organism, N -methyl- l -alloisoleucine and d -isoleucine substitute for N -methyl- l -valine and either d -valine or d -allo soleucine, respectively, in the antibiotic peptides.