The O-Carbamoyl-Transferase Alb15 Is Responsible for the Modification of Albicidin

Abstract

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.

Total Synthesis and Biological Assessment of Novel Albicidins Discovered by Mass Spectrometric Networking

“…Product ion spectra with collision‐induced dissociation (CID or HCD, shown in Figure S2, Table S1 in the SI) of albicidin typically rendered the formation of b‐ and y‐ion series. Apart from albicidin 1 ([M+H] + =843.262 Da) and carbamoyl albicidin 2 ([M+H] + =886.268 Da), we were able to propose structures of eight putative albicidin derivatives (Figure and SI): for propionyl‐albicidin 3 ([M+H] + =739.236 Da), a propionyl residue replaces 2‐methylcoumaric acid (MCA) and methoxy‐MCA‐albicidin in the case of 4 ([M+H] + =857.278 Da) is a methylated albicidin (+14.016 Da). Two further derivatives ([M+H] + =827.267 Da) bear 4‐amino‐3‐methoxybenzoic acid ( p AMBA) instead of the tetrasubstituted 4‐amino‐2‐hydroxy‐3‐methoxybenzoic acid ( p AMHBA) at building blocks E ( 5 ) and F ( 6 ).…”

“…Product ion spectra with collision-induced dissociation (CID or HCD, shown in FigureS2, Table S1 in the SI) of albicidin typically rendered the formation of b-and y-ion series. Apart from albicidin 1 ([M + H] + = 843.262 Da) [4] and carbamoyla lbicidin 2 ([M + H] + = 886.268 Da), [13] we were able to proposes tructures of eight putative albicidin derivatives (Figure 2a To unambiguously confirm the structures of the eight new derivatives of albicidin, at otal synthesis was envisaged.T he main challenge was the syntheses of the b-OMe-Cya-and of the b-OMe-Asn-albicidins.W ed ecided to synthesize both enantiomerso fl-b-OMe-Asn, because the stereoconfiguration at the a-position was already known, [6] whereas the configuration at the b-position introduced by the b-hydroxylating oxy-genaseAlb08 was unclear.…”

“…b) Subnetwork of albicidin and its derivatives.Black numbersindicates annotated spectra with ap utativec hemicalstructure,b lue numberss howsspectra which originate from sodium adducts. Nodes labelled with grey number representeither MS/MSs pectra of 13 Ci sotope peaks or of unknown compounds.…”

Synthesis of Albicidin Derivatives: Assessing the Role of N‐terminal Acylation on the Antibacterial Activity

“…Recently some of our studies in structure elucidation revealed another naturally occurring albicidin with an O‐carbamoylation on the para ‐coumaric acid. This modification enhances the activity to the lower nanomolar range …”

Entdeckung und Totalsynthese von natürlichen Cystobactamid‐Derivaten mit herausragender Aktivität gegen Gram‐negative Pathogene

“…Fürs ynthetisch hergestellte Albicidine [15][16][17][18] wurden SAR-Studien verçffentlicht, die zeigen, dass Va riationen am zentralen Linker [17] sowie am N-Terminus [18] zu Derivaten mit gleichbleibender oder verbesserter inhibi- Fürs ynthetisch hergestellte Albicidine [15][16][17][18] wurden SAR-Studien verçffentlicht, die zeigen, dass Va riationen am zentralen Linker [17] sowie am N-Terminus [18] zu Derivaten mit gleichbleibender oder verbesserter inhibi- …”