Abstract
Actinomycin synthetase I (ACMS I) activates 4-methyl-3-hydroxyanthranilic acid, the precursor of the chromophoric moiety of the actinomycin, as adenylate. The gene acmA of ACMS I was identified upstream of the genes acmB and acmC encoding the two peptide synthetases ACMS II and ACMS III, respectively, which assemble the pentapeptide lactone rings of the antibiotic. Sequence analysis and expression of acmA in Streptomyces lividans as enzymatically active hexa-His-fusion confirmed the acmA gene product to be ACMS I. An open reading frame of 234 base pairs (acmD), which encodes a 78-amino acid protein with similarity to various acyl carrier proteins, is located downstream of acmA. The acmD gene was expressed in Escherichia coli as hexa-His-fusion protein (Acm acyl carrier protein (AcmACP)). ACMS I in the presence of ATP acylated the purified AcmACP with radioactive p-toluic acid, used as substrate in place of 4-MHA. Only 10% of the AcmACP from E. coli was acylated, suggesting insufficient modification with 4'-phosphopantetheine cofactor. Incubation of this AcmACP with a holo-ACP synthase and coenzyme A quantitatively established the holo-form of AcmACP. Enzyme assays in the presence of ACMS II showed that toluyl-AcmACP directly acylated the thioester-bound threonine on ACMS II. Thus, AcmACP is a 4-MHA carrier protein in the peptide chain initiation of actinomycin synthesis.
Highlights
The actinomycins, a family of bicyclic chromopeptide lactones with strong antineoplastic activity [1], are produced by various streptomycete strains [2, 3]
4-methyl-3-hydroxyanthranilic acid (4-MHA), the ultimate amino-terminal residue of the 4-MHA pentapeptide lactone sequence, is activated by a separate adenylating enzyme, Actinomycin synthetase I (ACMS I) [10]. It has a size of 45 kDa and can adenylate a variety of benzene carboxylic acids structurally related to 4-MHA, e.g. 4-methyl-3-hydroxybenzoic acid (4-MHB) or p-toluic acid [10, 11]
The 5Ј-end of this open reading frame (ORF) encodes an amino acid sequence that is identical to the aminoterminal protein sequence of ACMS I, previously determined by microsequencing of the protein [13]
Summary
4-MHA, 4-methyl-3-hydroxyanthranilic acid; 4-MHB, 4-methyl-3-hydroxybenzoic acid; ACMS, actinomycin synthetase; ACP, acyl carrier protein; ORF, open reading frame; PMSF, phenylmethylsulfonyl fluoride; DTE, 1,4-dithiothreitol; PAGE, poly-. 4-MHA, the ultimate amino-terminal residue of the 4-MHA pentapeptide lactone sequence, is activated by a separate adenylating enzyme, ACMS I [10] It has a size of 45 kDa and can adenylate a variety of benzene carboxylic acids structurally related to 4-MHA, e.g. 4-methyl-3-hydroxybenzoic acid (4-MHB) or p-toluic acid [10, 11]. ACMS II catalyzed, albeit with lower efficiency than in the latter cases, formation of p-toluyl-threonine from threonine and chemically synthesized p-toluyl-adenylate This suggested that ACMS II, which cannot activate 4-MHA, would possess a binding domain with a 4Ј-phosphopantetheine cofactor (ACP domain) as an acceptor site for 4-MHA [8]. Cloning of the genes of ACMS II and III (acmB and acmC) showed that they lie closely linked in tandem on the chromosome of S. chrysomallus [13] Their analysis revealed an organization of the ACMSs into two and three modules, respectively, necessary for pentapeptide lactone assembly. We found directly downstream of the gene of ACMS I a small open reading frame (ORF) encoding a small ACP (acmD), which is the protein harboring the missing third 4Ј-phosphopantetheine cofactor required for condensation of 4-MHA with threonine on ACMS II
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