Actinomycin chromophore

Katz E, Weissbach H. Biosynthesb of the actinomycin chromophore Enzymatic conveisiort of 4-methy] 3 hydtoxyanchranilic acid to actinocin. J Biol Chem 1%2 237 882-886. 

Cmovcic, I., Siissmuth, R., and Keller, U. (2010) Aromatic C-methyltransferases with antipodal stereoselectivity for structurally diverse phenolic amino acids catalyze the methylation step in the biosynthesis of the actinomycin chromophore. Biochemistry, 49, 9698 9705. 

Evidence obtained with radioisotopically labeled tryptophan as well as with certain tryptophan analogues have established that tryptophan is a precursor of the actinomycin chromophore (Katz, 196O Sivak, Meloni, Nobili and Katz, 1962 Sivak and Katz, I962). In experiments with benzene ring-labeled tryptophan it was shown that the C-label of the amino acid was efficiently (11.7%) and preferentially incorporated into the phenoxazinone moiety of actinomycin (Table 3). The chromophore derivatives, actinocinin and desaminoactinocylthreonine dimethyl ester, obtained from actinomycin by acid hydrolysis (Brockmann and Grone,... 

By contrast, the amino acids of actinomycin were found to be devoid of radioactivity. In recent studies it has been found that incorporation of the C-label of benzene ring labeled tryptophan into actinomycin can be exceptionally high. During optimum synthesis as much as 25 to 35% of the radioisotope supplied is introduced into the actinomycin chromophore. Tryptophan, labeled in the alanine side chain, is not incorporated into the actinomycin chromophore to any significant extent (unpublished observations) suggesting that the benzenoid moiety of tryptophan is the precursor of the actinomycin chro-... 

The precursor function of tr tophan in the biosynthesis of the actinomycin chromophore was also demonstrated by experiments with certain methylated analogues of tryptophan (Sivak and Katz, 1962). Actinomycin S5mthesis by growing cells and washed suspensions of the organism was inhibited by a-, 4-, 5-, and 6-methyl-DL-tr5 tophan the order of their effectiveness proved to be a->4->5->6-methyl-DL-tryptophan. The inhibition of s mthesis observed with a-methyl and 4-methyltr tophan was examined in further detail and, it was possible to demonstrate that L-tr tophan (Fig. 5) and L-kynurenine were able to reverse the inhibition due to these analogues. 3-Hydroxy-DL-kynurenine and 3-hydroxyanthranilic acid were effective also in reversing the inhibition by... 

Birch (1958) had suggested that acetate rather than tryptophan may play an important role for s mthesis of the actinomycin chromophore. He postulated that the 3-hydroxy-4-methylanthranilic acid precursor might arise from four acetate molcules through a process of oxidation, reduction and amination. If acetate constituted the source of the anthranilate precursor, eight moles of acetate would be required for synthesis of one mole of the actinomycin chromophore and it would be expected that the actinocin formed would possess appreciable C-label. The results of an experiment with sodium acetate-2- C indicate that acetate is not directly involved as a precursor of the actinomycin chromophore (Sivak, Meloni, Nobili and Katz, I962). Of the total radioactivity incorporated into actinomycin by 5. antihioticus following addition of acetate- C, only 4% was present in the chromophore. On the other hand, the amino acid, proline, in the peptide portion of the actinomycin molecule possessed appreciable radioactivity. 

Table 6. Incorporation of L-methionine- CH into actinomycin chromophore synthesized...

The enzymatic synthesis of the actinomycin chromophore also has been described (Weissbach and Katz, 1961 Katz and Weissbach 1962 Salzman, Weissbach and Katz, unpublished results). An enzyme, designated phenoxa-zinone synthetase, was obtained from sonic extracts of S. antihioticus and was shown to catalyze the oxidative condensation of o-aminophenols to the corresponding phenoxazinone. When 3-hydroxy-4-methylanthranilic acid (4MHAA) was used as substrate, actinocin was synthesized. The enzyme has been purified approximately 25- to 30-fold from sonic or French pressure cell extracts of the organism by means of standard protein fractionation procedures (ammonium... 

Brockmann, H., u. H. Muxfeldt Konstitution und Synthese des Actinomycin Chromophors. Angew. Chem. 68, 69 (1956b). 

Katz, E., and H. Weissbach Biosynthesis of the actinomycin chromophore Enzymatic conversion of 4-methyl-3-hydroxyanthranilic acid to actinocin. J. Biol. Chem. 237, 882 (1962a). 

SiVAK, A., and E. Katz Biosynthesis of the actinomycin chromophore. Influence of a-, 4-, 5 and 6-methyl-DL-tryptophan on actinomycin synthesis. Biochim. et Biophys. Acta 62, 80 (1962). 

Almost all actinomycins have the same chromophore, a planar phenoxa2inone dicarboxyUc acid called actinocin. In dactinomycin, the stmcture of which is shown in Figure 12, the two pendent pentapeptide lactones are identical, but in other actinomycins these lactones may be different. In other actinomycins the first amino acid, amide linked with actinocin, is usually L-threonine, as in dactinomycin the second position is sometimes D-aHo-isoleucine instead of D-valine the third position may be sarcosine or oxoproline the fourth position is sarcosine and the fifth position is sometimes /V-methyl isoleucine instead of /V-methylvaline. The lactone ring is always present. 

C62H85BrNi2Oie,2C1oH13N504-11 H20 7-BromoactinomycinDbis(2 -deoxyguanosine), undecahydrate (BRAXGU)306 P212121 Z = 4 Dx = 1.36 R = 0.094 for 4000 intensities. The co-crystals contain one actinomycin D (chromophore part, only, is shown in the... 

Aromatization can also be accomplished by oxidative cascade reactions-a strategy often utilized by natural product biosynthesis [33]. For example, the phenoxazinone chromophore of actinomycin was proposed to be generated by a six-electron oxidation of aminophenols catalyzed by a copper-containing oxidase (Scheme 7.8) [34]. 

The first antitumor antibiotic was actinomycin A which was isolated from a Streptomyces species. The actinomycins are chromopeptides containing a planar chromophore, responsible for the bright color of the compounds, with peptide side chains. The most important representative of this group which is in clinical use is actinomycin D, or dactinomycin. 

The actinomycins, which are also produced by Streptomyces, not only kill bacteria but also are among the most potent antitumor agents known.b However, because of their extreme toxicity they are of little medicinal value. Actinomycin D, which is a specific inhibitor of RNA polymerase, contains a planar phenoxazone chromophore bearing two carboxyl groups, each one linked to an identical cyclic peptide made up of L-threonine, D-valine, L-proline, sarcosine (N-methylglycine), and L-methylvaline. 

Amino-4,6-dimethyl-3-oxo-3//-phenoxazine-l,9-dicarboxylic acid also named actinocin is the chromophor of the red antineoplastic chromopeptide actinomycin D (formula A). Two cyclopenta-peptide lactone rings (amino acids L-threonine, D-valine, L-proline, sarcosine, and A-methyl-L-valine) are attached to the carboxy carbons of actinocin by two amide bonds involving the amino groups of threonine. 

The chromophores of some of the peptide antibiotics may also be synthesized from amino acids. Thus the phenoxazine ring of actinomycin originates from tryptophan . 

The blue oxidases-related enzymes include phenoxazinone synthase from S. antibioticus This enzyme is a copper-containing oxidase that catalyzes the coupling of 2-aminophenols to form the 2-aminophenoxazinone chromophore. This reaction constitutes the final step in the biosynthesis of the potent antineoplastic agent actinomycin. The crystal structure of the oxidized form phenoxazinone synthase from S. anibioticus has been determined. It has been solved in his hexameric form. One monomer is very similar to Iaccase or ascorbate oxidase but it contains a long loop, which connects two domains and stabilizes the hexameric structure. Bound... 

Actinomycins are chromopeptides, generally containing the same chro-mophore, based on the tricyclic system of phenoxazinone (phenoxazone), and known as actinocin, Fig. (8). The chromophore is responsible for the intense yellow, orange or red colours of actinomycins. The first known actinomycin was isolated from a culture medium of an actinomyces, Streptomyces sp. [174], and caused some interest on account of its antibiotic activity. Many other actinomycins were isolated later and characterised by their antibiotic and/or antitumor activities [175]. Among these, the best known is actinomycin D (Dactinomycin), Fig. (8). 

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]. 

Actinomycins, peptide antibiotics produced by various strains of Streptomyces. Actinomycins are orange-red bacteriostatic and cytostatic, but highly toxic, chromopeptides. The chromophore actinocin,... 

Figure 8. Actinomycin with two identical 16-membered cyclic pentapeptide rings attached to a chromophore. Each ring has two cis amide linkages occurring between Sar-LPro and LPro-oVal (Jain and Sobell, 1972).

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