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

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Actinomycins consist of two pentapeptide lactone rings attached to 2-amino-4,6-dimethyl-3-oxo-phenoxazine-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 β-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.DFG, EXC 314, Unifying Concepts in Catalysi

The O-carbamoyl-transferase Alb15 is responsible for the modification of albicidin

Albicidin is a potent antibiotic and phytotoxin produced by Xanthomonas albilineans which targets the plant and bacterial DNA gyrase. We now report on a new albicidin derivative which is carbamoylated at the N-terminal coumaric acid by the action of the ATP-dependent O-carbamoyltransferase Alb15, present in the albicidin (alb) gene cluster. Carbamoyl-albicidin was characterized by tandem mass spectrometry from cultures of a Xanthomonas overproducer strain and the gene function confirmed by gene inactivation of alb15 in X. albilineans. Expression of alb15 in Escherichia coli and in vitro reconstitution of the carbamoyltransferase activity confirmed albicidin as the substrate. The chemical synthesis of carbamoyl-albicidin finally enabled us to assess its bioactivity by means of in vitro gyrase inhibition and antibacterial assays. Compared to albicidin, carbamoyl-albicidin showed a significantly higher inhibitory efficiency against bacterial gyrase (∼8 vs 49 nM), which identifies the carbamoyl group as an important structural feature of albicidin maturation. (Résumé d'auteur

The <i>O</i>-Carbamoyl-Transferase Alb15 Is Responsible for the Modification of Albicidin

Albicidin is a potent antibiotic and phytotoxin produced by <i>Xanthomonas albilineans</i> which targets the plant and bacterial DNA gyrase. We now report on a new albicidin derivative which is carbamoylated at the <i>N</i>-terminal coumaric acid by the action of the ATP-dependent <i>O</i>-carbamoyltransferase Alb15, present in the albicidin (<i>alb</i>) gene cluster. Carbamoyl-albicidin was characterized by tandem mass spectrometry from cultures of a <i>Xanthomonas</i> overproducer strain and the gene function confirmed by gene inactivation of <i>alb15</i> in <i>X. albilineans</i>. Expression of <i>alb15</i> in <i>Escherichia coli</i> and <i>in vitro</i> reconstitution of the carbamoyltransferase activity confirmed albicidin as the substrate. The chemical synthesis of carbamoyl-albicidin finally enabled us to assess its bioactivity by means of <i>in vitro</i> gyrase inhibition and antibacterial assays. Compared to albicidin, carbamoyl-albicidin showed a significantly higher inhibitory efficiency against bacterial gyrase (∼8 vs 49 nM), which identifies the carbamoyl group as an important structural feature of albicidin maturation