Biosynthetic rivalry of o-aminophenol-carboxylic acids initiates production of hemi-actinomycins in Streptomyces antibioticus

Abstract

Actinomycins consist of two pentapeptide lactone rings attached to 2-amino-4,6-dimethyl-3-oxophenoxazine-1,9-dicarboxylic acid (actinocin). The actinocin moiety is formed through oxidative condensation of two 3-hydroxy-4-methylanthranilic acid (4-MHA) pentapeptide lactones (actinomycin halves) as the last step of actinomycin biosynthesis. We found that feeding of 4-MHA or its putative biogenetic precursor 3-hydroxyanthranilic acid (3-HA) to Streptomyces antibioticus induced formation of different new compounds at the expense of actinomycins. These contain only one pentapeptide lactone ring attached to the b-side of their phenoxazinone ring systems and are formed through premature condensation of the externally added abundant 4-MHA or 3-HA with actinomycin halves. They were termed hemi-actinomycins and C-demethyl-hemi-actinomycins, respectively, which differ from each other in the presence or absence of one or both methyl groups in their phenoxazinone moieties. 3-HA also induces synthesis of various C-demethylactinomycins formed through condensation of actinomycin halves in which 3-HA had been incorporated by the 4-MHA incorporating enzyme in lieu of 4-MHA. 3-HA was not converted to 4-MHA as revealed by its inability to stimulate synthesis of actinomycin or hemi-actinomycin synthesis and thus remained a substrate analogue of 4-MHA rather than its precursor.In contrast to S. antibioticus, actinomycin-producing streptomycetes such as Streptomyces chrysomallus or Streptomyces parvulus do not form hemi-structured actinomycins when fed with 3-HA or 4-MHA.They do not possess the enzyme phenoxazinone synthase (PHS) which in S. antibioticus is present and most probably catalyses premature condensation of abundant 4-MHA or 3-HA with actinomycin halves.Testing hemi-acinomycin IV for drug activity revealed that it intercalates into DNA and inhibits relaxation and supertwisting of DNA by topoisomerase I and DNA-gyrase like actinomycin IV (D). Moreover, it has inhibitory activity on growth of Bacillus subtilis.

MALDI‐TOF mass spectrometry, an efficient technique for in situ detection and characterization of actinomycins

“…The pathways for the production of the C‐demethyl derivatives and their biochemical characterization are described in the study of Crnovčić et al . For the hemi ‐actinomycins such data are presented in another recent paper …”

MolNetEnhancer: enhanced molecular networks by integrating metabolome mining and annotation tools

“…The annotation of this MF was guided by DEREPLICATOR and VarQuest annotations as well as the Mass2Motif that 10 of its members shared. We could annotate this Mass2Motif as the amino acid lactone loop present twice in actinomycins using reference data from literature [45]. The unique combination of four actinomycin-related mass fragments was only present in the 10 MF members, thereby reinforcing the DEREPLICATOR and VarQuest annotations.…”

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4‐Oxoproline Synthase

“…Accordingly, the proton in position 4 of the proline moiety gave a multiplet at δ =4.31 ppm, which was not seen in the spectrum of PPL 1, and this confirmed that the 4‐position of the proline ring was substituted. These compounds thus have nearly the same structures as natural Acm halves (Scheme ) but cannot react with each other to yield Acms and therefore accumulate in the cultures.…”

“…Strains and cultures : S. chrysomallus ATCC 11523, Streptomyces parvulus ATCC 12434, and S. antibioticus ATCC 14888 (IMRU3720) were maintained and grown as described previously . Mycelium used for biotransformation experiments was from 80 h old cultures (actively synthesizing Acms) of S. antibioticus grown in previously described chemically defined media .…”

“…Their diversity is based on amino‐acid substitutions in their pentapeptide lactone rings, whereas their chromophoric moiety, 2‐amino‐3‐oxo‐4,6‐dimethyl‐1,9‐phenoxazine‐dicarboxylic acid, is always the same . Biochemical data suggest that Acms such as the representative actinomycin D (Acm D), are formed in the last step of their biosynthesis through oxidative condensation (dimerization) of Acm halves . The latter consist of single pentapeptide lactone rings, to which 3‐hydroxy‐4‐methylanthranilic acid (4‐MHA) residues are attached, which react to the phenoxazinone (Scheme ).…”