Actinomycins methylation

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. 

Many NRPs such as cyclosporin, complestatin, actinomycin, and chondramide contain N-methyl amides. M-Methyl transferase (N-MT) domains utilize S-adenosylmethionine (SAM) as a cofactor to catalyze the transfer of the methyl group from SAM to the a-amine of an aminoacyl-S-PCP substrate. The presence of M-methylamides in NRPs is believed to protect the peptide from proteolysis. Interestingly, N-MT domains are incorporated into the A domains of C-A-MT-PCP modules, between two of the core motifs (A8 and A9). MT domains contain three sequence motifs important for catalysis. ° 0-Methyl transferase domains are also found in NRPSs and likewise use the SAM cofactor. For instance, cryptophycin and anabaenopeptilide synthetases contain 0-MT domains for the methylation of tyrosine side chains. These 0-MT domains lack one of the three core motifs described for N-MT domains. ... 

The induction of PAL activity at the onset of vascular differentiation can be shown by the use of plant tissue cultures (37-39). Xylem cells with secondary and lignified walls are differentiated over a time course of 3-14 days by the application of the plant growth factors naphthylene acetic acid (NAA) and kinetin in the ratio 5 1 (1.0 mg/liter NAA, 0.2 mg/liter kinetin) to tissue cultures of bean cells (Phaseolus vulgaris) (37,40). The time for differentiation varies with the type of culture, solid or suspension, and with the frequency and duration of subculture, but for any one culture it is relatively constant (37,41,42). At the time of differentiation when the xylem vessels form, the activity of PAL rises to a maximum. The rising phase of the enzyme activity was inhibited by actinomycin D and by D-2,4-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide (MDMP) applied under carefully controlled conditions (42). This indicated that both transcription and translation were necessary for the response to the hormones. Experiments using an antibody for PAL and a cDNA probe for the PAL-mRNA have also shown that there is an increase in the amount of transcript for PAL during the formation of lignin when Zinnia mesophyll cells are induced to form xylem elements in culture (Lin and Northcote, unpublished work). 

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 phenoxazinone ring system of the actinomycins (19 ) is apparently formed from kynurenine and 3-oxykynurenine with methylation taking place at a late stage 73. Reports on amino acid variation and the mechanism of biosynthesis of the D-amino acids present in the side chains continue to appear 71t>75,76 >77>78,... 

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]. 

Purification has been reported of phenoxazinone synthase, the enzyme which catalyses phenoxazinone formation from 4-methyl-3-hydroxyanthranilic acid (123) in the biosynthesis of actinomycin (124). Two forms of the enzyme, one of high and one of low molecular weight, were isolated. The relative amount of the... 

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). 

Aromatic methylation involves transfer of a methyl group from methionine. The mechanism of this transfer in actinomycin biosynthesis was examined by feeding [Me- H3]methionine to S. antibioticus The actinomycin isolated contained tri-and hexa-deuterio-species, from which it follows that all the hydrogens of the methyl... 

The remaining atoms in anthramycin (166) can be identified with 4-methyl-3-hydroxyanthranilic acid (171), an intermediate in the biosynthesis of actinomycins from tryptophan.In accord, [7a- C]tryptophan was well incorporated into anthramycin. " ... 

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). ... 

Under suitable Conditions, the protoplasts from Streptomyces anti-bioticus readily incorporated radioactively-labelled threonine, valine, proline, glycine and methionine into the relevant positions of actinomycins. Administration of radioactive tryptophan yielded actinomycins labelled in the chromophore. When the oxygen pressure was low, trace amounts of 4-methyl-3-hydroxy-anthranilic acid, which is an intermediate in the biosynthesis of the chromophoric part of actinomycin, could be detected. 

VaU,Pro ), N-me thyl(MeVal ), 8-hexadeuterol-actinomycin Ci [2(Val, Proa), N-methyl(MeVals) -pentadeuteroj-actinomycin Ci [2(VaU,, Pro .a),N-methyl(MeVaU,... 

Fig. 3. Chemical structure of actinomycin Cj (D). - Abbreviations MeVal, methyl-valine sar, sarcosine Pro, proline Val, valine Thr, threonine.

Keller and coworkers succeeded in the preparation of several aromatic carboxylic acid-activating starter enzymes involved in the biosynthesis of actinomycin (4-methyl-3-hydroxyanthranilic acid) (105,113,114), triostin (qninoxaline-2-carboxylic acid) (105,115), and mikamycin (3-hydroxypicolinic acid) (105,116). More recently, related enzymes have been identified in the Actinomycetes that form the thiopeptides nosihep-tide and thiostreptone (117,118), and in the pristinamyctn biosynthetic system (de Crecy-Lagard V, personal communication). Whether direct acylation of the pantetheine-attached starter amino acid occurs, or an additional transferase frinction is required, has not been settled. 

Kellei U. Schlumbohm W. Purification and characterization of actinomycin synthetase I. a 4-methyl-3-hydroxv-anthraniilic acid-AMP ligase from Streptomyces chrysomallus. J Biol Chem 1992,267 11745-11752. 

Figure 6 Assembly of 4-MHA and the five constituent amino acids in the synthesis of 4-MH.A pentapeptide lactone D. The conesponding actinomycin D arises by oxidative condensation of two of these 4-MHA pentapeptide lactone moieties, catalyzed by phenoxazinone synthase. Sar = sarcosine (N-methylglycine) MeVal M-methyl-L-valine.

Jones GH. Actinomycin synthesis in Streptomyces antibioticus Enzymatic conversion of 3-hydroxyanthranilic acid to 4.methyl 3 hydroxvanihranilic acid. J Bacteriol 1987 169 5575-5 578. 

Keller U, Kleinkauf H. Zocher R. 4 Methyl-3-hydroxyanthranilic acid (4 MHA) activating enzyme from actinomycin producing Streptumyces e/irysomallus. Biochem 1984 ... 

Weissbach H, Redfletd B, Beaven V, Katz E. 4-Methvl-3-hydroxyanthranilic acid, an intermediate in aciinomycin biosynthesis- Biochem Biophys Res Commun 1965 19 524-530-Keller U. Acyl pemapeptide lactone synthesis in actinomycin-producing streptomycetes by feeding with structural analogs of 4 methyl 3.hydroxyamhranilk acid (4-MMA). J Biol Chem 1984 S9i8226-8231. 

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