Actinomycins synthetase

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. ... 

Like modular PKSs, peptide synthetases also epimerize some substrates and/or intermediates. For example, the starter substrate amino acid of cyclosporin A is D-Ala. Racemization of alanine is not catalyzed by an integrated subunit of cyclosporin A synthetase, but by alanine racemase. This is a separate, pyridoxal phosphate-dependent enzyme [ 193]. In contrast, Grsl and Tycl covalently activate L-Phe as a thioester and subsequently epimerize the amino acid [194]. D-Phe is the only epimer accepted as a substrate for dipeptide formation by Grs2 and Tyc2 [195, 196]. No racemization activity is detected in a pantetheine-deficient mutant of Grsl [197]. Deletion mutagenesis pointed to the requirement of the COOH-terminal part of the module for epimerizing L-Phe to D-Phe [180]. In contrast, the biosynthesis of actinomycin D, a bicyclic chromo-pentapeptide lactone (Fig. 10), involves formation of the dipeptide 6-MHA (methylanthranilic acid)-L-Thr-L-Val prior to epimerization of the L-Val exten-... 

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]. 

The pentapeptide moiety is not attached to the cromophore until acti-nocin is formed. In Str. chrysomallus, the whole enzyme complex responsible for the modification and polymerisation of the aminoacids which constitute the peptide chain has been isolated and characterised as being composed of three fractions (actinomycin synthetase I, II, and III) [183-184]. 

In table 1 the present state of characterization of several enzyme systems discussed in this publication is shown. While synthetases for gramicidin S, alamethicin or enniatin are available in purified or partially purified forms, the biosynthesis of actinomycins and valinomycin cannot yet be studied in vitro presumably,because the preparation of a cell free extract of these membrane bound systems needs more subtle and advanced techniques of membrane isolation and fragmentation. A promising approach for the investigation of such systems has been found in the study of protoplasts from the producer organism of these antibiotics. 

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. 

From all data available so far on peptide and depsipeptide synthetases, it af >ears that enzymes of bacterial origin such as gramicidin 5 synthetase, tyrocidine synthetase, or actinomycin synthetase (all reviewed in Ref. 61) have subunit structure, whereas fungal enzymes such as SDZ 214-103 synthetase (62) (see Section VI.C), SDZ 90-215 synthetase (63), and cyclosporin synthetase consist of single polypeptide chains. Cyclospcmn synthetase represents the most complex one. Like type 1 polyketide synthases, these enzymes are designated multifunctional polypeptides the bacterial enzymes, on the other hand, are called "multienzyme complexes, analogous to type 11 polyketide synthases (64). 

Fawaz F, Jones CH. Actinomycin synthesis in Streptomyces ontibiotinu Purification and properties of hydroxYanthranilate-4 mechylcransfetase. ] Biol Chem 1988 263 4602-4606. Jones GH. Combined purification of actinomycin synthetase I and 3-hydroxyanthranilic acid 4-methylttansferase from Streptamyces aruibi rmcus. ] Biol Chem 1993 268 6831-6834. Keller U, Schlumbohm W. Purification and characterization of actinomycin synthetase I. a 4 methy -3-hydroxyanthranilic acid AMP ligase from Streptomjees chrysomallus. J Biol Chem 1992 267 11745-11752. 

Keller U- Actinomycin synthetases Multifunctional enzymes responsible (or the synthesis of the peptide chains of actinomycin. J Biol Chem 1987 262 5852-5856. 

Overall half-life for ALA-synthetase. After addition of actinomycin D, ALA-synthetase increased slightly for 2 hours then the activity remained at a plateau for 3 hours, and finally it decayed with ti/2 of 3 hours. In actinomycin D -h heme treatment, the decay of ALA-synthetase took place within 3 hours of drug addition. 

The addition of ferrie heme chloride at 5 /ig/ml of medium did not alfect general synthesis of protein, RNA, or DNA as determined with tracers [128]. Hemin did not directly affect the activity or lifetime of ALA-synthetase either in isolated mitochondria or in cell culture. When cells were cultured with AIA for 12 hours to increase the level of ALA-synthetase, the medium was changed, and both actinomycin D and hemin were added, the decay rate for ALA-synthetase was increased from i/2=5.2 hours (for actinomycin D alone) to t,/2=3.6 hours (Table III). These results suggest that hemin either shortened the lifetime of mRNA for ALA-synthetase or interfered specifically with the synthesis of ALA-synthetase. Previous studies had shown that heme did not stimulate ribonuclease activity. The inhibitory effect of hemin on the transport of soluble ALA-synthetase into the mitochondria has been discussed in Section V. 

The synthesis of the enzyme that makes ALA was not blocked by actinomycin D or other inhibitors of RNA synthesis. This result suggests that there may be suflicient of various RNA compounds already stored in the proplastid to be nonlimiting during the first 6 to 12 hours of illumination to serve for the synthesis of ALA-synthetase. If inhibitors of RNA synthesis are efiective in these cells, then the effect of illumination may be to bring about the production of the limiting enzyme for ALA synthesis by activating the translation rather than at the transeription step. A summary of the hypotheses on the control mechanism for CHL synthesis is presented in Fig. 11. 

Effect of actinomyin JD, cycloheximide, and azacytidine on de novo purine biosynthesis Table 2 illustrates that all three inhibitors, actinomycin D, cycloheximide, and azacytidine inhibit de novo purine biosynthesis as measured by the incorporation of -formate into total purines Labelling was for 2 hours, and the results are the average of three experiments Controls had between 25,000 and 30,000 cpm/10 cells Actinomycin D had no effect on Li vitro assayed PRPP amidotransferase or PRPP synthetase activities for cell pretreated for 2 hours with the drug ... 

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