Actinomycins 7-13- precursor

Actinomycin.—Kynurenine and 3-hydroxykynurenine are actinomycin precursors in Streptomyces antibioticus (cf. Vol. 6, p. 42). Recently, kynureninase and hydroxykynureninase activity has been identified in S. parvulus cultures and the latter activity was found to show correlation with actinomycin formation.57... 

The thiol-template mechanism is utilized in other enzymes involved in production of peptide-based antibiotics. Actinomycin synthetase II (ACMSII) and b-L-(a-aminoadipolyl)-L-cysteinyl-D-valine (ACV) synthetase catalyze the stereoinversion of valine residues vithin peptide-based antibiotics, and employ ATP and the PAN cofactor in a mechanism similar to that depicted in Fig. 7.14 [58, 59]. ACMSII catalyzes the stereoinversion of a valine within the tripeptide 4-MHA-L-Thr-D-Val (MHA, 4-methyl-3-hydroxyanthranilic acid), which is a precursor for the antibiotic actinomycin D. ACV synthetase catalyzes the stereoinversion of the valine within ACV, which is a precursor for penicillin and cephalosporin [60-63]. ACV synthetase has been shown to have much broader substrate specificity, also accepting non-natural substrates [64, 65]. 

Little is known concerning the mechanism of biogenesis of the often occurring Y-methylamino acids. There is evidence that glycine and l-valine are the direct precursors of sarcosine > and Y-methyl-L-valine in actinomycin biosynthesis and that the methyl group is provided by methionine . 

Actinomydn.—The phenoxazinone skeleton of the actinomycins (181) has been shown to derive from two molecules of tryptophan via 3-hydroxy-4-methylanthranilic acid (185),methionine serving as the source of the aromatic methyl groups.Depression of the incorporation of radioactive tryptophan into actinomycin by kynurenine (182) and 3-hydroxy-4-methylkynurenine (184) indicated that (182) and (184) could well be biosynthetic intermediates. In contrast 3-hydroxykynurenine (183) was found to have little or no effect on tryptophan incorporation, thus casting doubt on its participation in actinomycin biosynthesis. However, both [ Hjkynurenine [as (182)] and [ H]-3-hydroxykynurenine [as (183)] have recently been shown to be efficient precursors for actinomycin in Streptomyces antibioticus. 3-Hydroxykynurenine (183) is therefore implicated in actinomycin biosynthesis and further substantive evidence is provided for the pathway tryptophan —> (182) — (183) — (184) —> (185) —> actinomycin (181). 

Naturally-occurring actinomycins show as a common feature the presence of the phenoxazinone chromophore, doubly-linked to a pentapeptide which forms a macrocyclic structure. Differences among actinomycins arise from variations in composition of the pentapeptide. The precursors of the aminoacids, forming the pentapeptide, have been singled out [176-178]. 

Actinomydn.—4-Methyl-3-hydroxyanthranilic acid (131) is an important precursor for actinomycin (132) cf. ref. 6. Further support for its role as a biosynthetic intermediate is its accumulation in mutants of Streptomyces parvulus that are unable to synthesize (132). ... 

Infection with picornaviruses results in a strong (though selective) inhibition of the cellular ENA synthesis (5, 4> 85> 84), and the appearance of a new, virus-induced ENA synthesizing activity. The latter was soon shown to be insensitive to the action of Actinomycin D (5), an antibiotic which prevent DNA-dependent ENA synthesis by intercalating in the dG-dC sequences of the template MA (6, 7) These observations suggested the possibility of treating infected cultures with the antibiotic in order to suppress host-cell activity and measxiring the incorporation of radioactive precursors into viral ENA. Since the precursors added to the medium do not equilibrate instantaneously with the intracellular pool of nucleotides, a correction for this fact must be introduced, at least for the very early times. 

At the biochemical level, the development of a cell-free system capable of synthesizing actinomycin from the relevant precursors remains to be achieved. In the absence of such... 

Hanada M, Sugawara K, Nishiyama Y, Kamei H. Haiori M, Konishi M. Protactin, a new metabolite and a possible precursor of the actinomycins. J Antibiot 1992 45 20-28. 

Mason KT, Shaw GJ. Kati E. Biosynthetic studies with L-[2, J- Hj]valine as precursor of the D-valine moiety in actinomycin. Arch Biochem Biophys 1977 180 509-513. 

Studies on the incorporation of labeled amino acids in thyroid slices have provided a more detailed description of the mechanism of synthesis of the hormone. The radioactivity first appears in soluble polypeptides with sedimentation coefficients of 3, 8, or 12. Puromy-cin or actinomycin blocks the incorporation of the precursor into the soluble polypeptides. The half-life of the messenger RNA for thyroglobulin polypeptide was estimated to be 15-20 hours. Indeed, after inclusion of actinomycin in the incubation mixture, thyroglobulin synthesis continues for several hours. The subunits are transferred from the site of synthesis to an assembly center, in which the subunits are iodinated, carbohydrate units are included in the molecule, and subunits are condensed into a finished protein. Puromycin fails to interfere with the formation of 19 S units. 

Katz has reported that D-valine inhibits actinomycin synthesis and suggested that L-valine is the precursor of the D-valine which may occur in the molecule at sites B and B. ... 

Certain D-amino acids can be formed in bacteria by processes involving a racemase and D-amino acid transaminases. Amino acid activating systems have been found for D-alanine in Staph, aureus and a number of other bac-teria 34 is thus possible that free D-amino acids are sometimes incorporated as such into peptide antibiotics. On the other hand, the D-valine fragment of the penicillins arises from changes in an intermediate peptide that contains an L-valine residue. L-Valine in the culture fluid appears to be the precursor of the D-valine residue found in some of the actinomycins, but this relationship does not reveal the stage at which the inversion of configuration occurs. Differences in the permeability of cells or intracellular structures to l- and D-amino acids are liable to complicate the interpretation of experiments in this field. 

On the basis of the early observation that the increases in microsomal enzyme activity produced by phenobarbital and 3-methylcholanthrene were blocked by actinomycin-D, it was suggested that enzyme induction resulted from the synthesis of new enzyme protein which was, in turn, dependent upon the DNA-directed synthesis of a messenger-like RNA. Treatment of rats with 3-methylcholanthrene causes an increase of about 40% in the level of RNA in rat liver nuclei and the nuclear RNA from 3-methylcholanthrene-treated rats is more active in directing protein synthesis than RNA from control animals. Moreover, the in vitro incorporation of radioactive precursors such as orotic acid or cytidine triphosphate into nuclear RNA is 50 to 100% greater in preparations from 3-methylcholanthrene-treated animals than controls. It is of interest that treatment of rats with phenobarbital has been recently reported to result in a marked suppression of endogenous hepatic ribonuclease activity. 

Figure 9.19. QITESI-MS/MS spectraofthe [M - 5H] complex containing duplex d(GCGGGGATGGGGCG)/ (CGCCCCATCCCCGC) and actinomycin by (a) CAD (0.86 V, 30 ms) (73%) and (b) IRMPD (50 W, 1.5 ms) (6%) with the portion of uninformative base loss ions given in parentheses. Fragment labels include a subscript G or C if they pertain specifically to the G-rich or C-rich strand of the duplex, respectively. The magnification scale applies to both spectra. Precursor ion is denoted with an asterisk ( ). (Reprinted with permission from Wilson, J. J. Brodbelt, J. S. Anal. Chem. 2007, 79, 2067.)...

More direct evidence that polymyxin synthesis proceeds by a mechanism that differs from that of protein synthesis comes from experiments with growing cultures of B. polymyxa in which the effects of inhibitors of protein and nucleic acid synthesis on the incorporation of radioactive precursors into protein and polymyxin B were studied (Paulus and Gray, 1964). As shown in Table 7, chloramphenicol, actinomycin D, and to a lesser extent puromycin inhibit the incorporation of L-threonine- C into protein but stimulate its incorporation into polymyxin B. This differential effect of inhibitors on protein and polymyxin synthesis strongly supports the hypothesis that polymyxin synthesis does not proceed by the kind of template mechanism that operates in protein synthesis. 

Controlled biosynthesis can be defined as the technique of predetermining the structure of a new antibiotic by furnishing specific chemical precursors to the antibiotic-producing microorganism (Woodruff and McDaniel, 1958). This technique has been successfully applied to the formation of modified forms of actinomycin. 

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