A Six‐Oxidase Cascade for Tandem C−H Bond Activation Revealed by Reconstitution of Bicyclomycin Biosynthesis

Abstract

As a commercial antibiotic, bicyclomycin (BCM) is currently the only known natural product targeting the transcription termination factor rho. It belongs to a family of highly functionalized diketopiperazine (DKP) alkaloids and bears a unique O-bridged bicyclo[4.2.2]piperazinedione ring system, a C1 triol, and terminal exo-methylene groups. We have identified and characterized the BCM biosynthetic pathway by heterologous biotransformations, in vitro biochemical assays, and one-pot enzymatic synthesis. A tRNA-dependent cyclodipeptide synthase guides the heterodimerization of leucine and isoleucine to afford the DKP precursor; subsequently, six redox enzymes, including five α-ketoglutarate/Fe -dependent dioxygenases and one cytochrome P450 monooxygenase, regio- and stereoselectively install four hydroxy groups (primary, secondary, and two tertiary), an exo-methylene moiety, and a medium-sized bridged ring through the functionalization of eight unactivated C-H bonds.

Genome mining of cyclodipeptide synthases unravels unusual tRNA-dependent diketopiperazine-terpene biosynthetic machinery

“…Modification of the heterocycle or modification of amino acid side chains of the constituent amino acids can therefore be expected. However, so far only four CDPS-dependent assembly lines have been fully elucidated, with modifications such as oxidation 16 , 18 , 20 , and methylation 18 . In this study, we were able to demonstrate that dmt1 encodes CDPS-dependent machinery involved in DMT biosynthesis both in vivo and in vitro.…”

“…As a matter of fact, CDPSs generally occur associated with tailoring enzymes 15 . Nonetheless only four CDPS-dependent pathways have been fully understood so far, including albonoursin 7 , 16 , nocazines 17 , 18 , pulcherriminic acid 19 , and bicyclomycin 20 , 21 . Compared to the canonic NRPS pathways, CDPS pathways are still underexplored.…”

Unrivalled diversity: the many roles and reactions of bacterial cytochromes P450 in secondary metabolism

“…287 One of the more impressive series of transformations reported to occur during bacterial DKP synthesis is found in the biosynthesis of bicyclomycin. 293,294 In this pathway, the initial cyclo(L-Leu-L-Ile) DKP undergoes six oxidation events (catalysed by ve a-ketoglutarate/Fe 2+ -dependant dioxygenases (BcmB/C/E/F/G) and one cytochrome P450 (BcmD)) to afford the nal, bicyclic structure of bicyclomycin. Within this pathway, the BcmD has been identied as carrying out the penultimate oxidation reaction, which affords hydroxylation of the C6 position of the DKP ring (Fig.…”

“…24C). 294 Thus, DKP-modication by cytochrome P450s -whilst relatively rare in bacteria -provides several examples of novel P450-catalysed transformations and such transformations should always be considered when P450s are identied in close proximity to a cyclodipeptide synthase.…”

A Promiscuous Cytochrome P450 Hydroxylates Aliphatic and Aromatic C−H Bonds of Aromatic 2,5‐Diketopiperazines

“…The DKP core is a privileged scaffold in drug research, and selective hydroxylation of the core by chemical methods is lengthy and sometimes synthetically challenging . So far, only three P450s, CYP121, CYP134A1, and BcmD, are known to be involved in the biosynthesis of DKP natural products mycocyclosin, pulcherriminic acid, and bicyclomycin, respectively (Scheme A).…”