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

Meng, Song; Han, Wei; Zhao, Juan; Jian, Xiao‐Hong; Pan, Hai‐Xue; Tang, Gong‐Li

doi:10.1002/ange.201710529

Cited by 24 publications

(33 citation statements)

References 47 publications

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“…This unambiguously confirmed that this was the BCM biosynthetic gene cluster. Our result agrees with those of recent studies by Patteson et al ( 32 ) and Meng et al ( 33 ) who, in parallel with our study, have reconstituted in vitro the functions of the CDPS and the oxidative steps in the S. cinnamoneus pathway.…”

Section: Resultssupporting

confidence: 94%

Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes

Vior

Lacret

Chandra

et al. 2018

Appl Environ Microbiol

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Bicyclomycin (BCM) is a clinically promising antibiotic that is biosynthesized by Streptomyces cinnamoneus DSM 41675. BCM is structurally characterized by a core cyclo(l-Ile-l-Leu) 2,5-diketopiperazine (DKP) that is extensively oxidized. Here, we identify the BCM biosynthetic gene cluster, which shows that the core of BCM is biosynthesized by a cyclodipeptide synthase, and the oxidative modifications are introduced by five 2-oxoglutarate-dependent dioxygenases and one cytochrome P450 monooxygenase. The discovery of the gene cluster enabled the identification of BCM pathways encoded by the genomes of hundreds of Pseudomonas aeruginosa isolates distributed globally, and heterologous expression of the pathway from P. aeruginosa SCV20265 demonstrated that the product is chemically identical to BCM produced by S. cinnamoneus. Overall, putative BCM gene clusters have been found in at least seven genera spanning Actinobacteria and Proteobacteria (Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria). This represents a rare example of horizontal gene transfer of an intact biosynthetic gene cluster across such distantly related bacteria, and we show that these gene clusters are almost always associated with mobile genetic elements.IMPORTANCE Bicyclomycin is the only natural product antibiotic that selectively inhibits the transcription termination factor Rho. This mechanism of action, combined with its proven biological safety and its activity against clinically relevant Gram-negative bacterial pathogens, makes it a very promising antibiotic candidate. Here, we report the identification of the bicyclomycin biosynthetic gene cluster in the known bicyclomycin-producing organism Streptomyces cinnamoneus, which will enable the engineered production of new bicyclomycin derivatives. The identification of this gene cluster also led to the discovery of hundreds of bicyclomycin pathways encoded in highly diverse bacteria, including in the opportunistic pathogen Pseudomonas aeruginosa. This wide distribution of a complex biosynthetic pathway is very unusual and provides an insight into how a pathway for an antibiotic can be transferred between diverse bacteria.

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Section: Resultssupporting

confidence: 94%

Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes

Vior

Lacret

Chandra

et al. 2018

Appl Environ Microbiol

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“…NRPSs, for example, are traditionally considered to be nature's go-to machinery for catalysing amide bond formation independently of the ribosome during NP biosynthesis, but in recent years, many pathways have been characterized that employ alternative strategies for assembling amino and carboxylic acid substrates 182,183 . Examples include a transglutaminase homologue that catalyses N-acyl-β-peptide link formation during andrimid (36) biosynthesis in various γ-proteobacterial species 184 , the use of ATP-grasp enzymes such as that involved in the assembly of the β-lactone belactosin (37) 185 and cyclodipeptide synthases that utilize activated aminoacyl-tRNAs to biosynthesize diketopiperazine NPs, such as bicyclomycin (38) [186][187][188] . Indolmycin (39) is a potent antibacterial compound produced by various bacteria that is derived from the condensation of two nonproteinogenic amino acids, indolmycenic acid and D-4,5-dehydroarginine.…”

Section: Pathways Lacking Signature Biosynthetic Genesmentioning

confidence: 99%

The hidden enzymology of bacterial natural product biosynthesis

Scott

Piel

2019

Nat Rev Chem

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Bacterial natural products display astounding structural diversity, which, in turn, endows them with a remarkable range of biological activities that are of significant value to modern society. Such structural features are generated by biosynthetic enzymes that construct core scaffolds or perform peripheral modifications, and can thus define natural product families, introduce pharmacophores and permit metabolic diversification. Modern genomics approaches have greatly enhanced our ability to access and characterize natural product pathways via sequence-similaritybased bioinformatics discovery strategies. However, many biosynthetic enzymes catalyse exceptional, unprecedented transformations that continue to defy functional prediction and remain hidden from us in bacterial (meta)genomic sequence data. In this Review, we highlight exciting examples of unusual enzymology that have been uncovered recently in the context of natural product biosynthesis. These suggest that much of the natural product diversity, including entire substance classes, awaits discovery. New approaches to lift the veil on the cryptic chemistries of the natural product universe are also discussed. Bacterial natural products (NPs) are specialized metabolites that encompass an extraordinary breadth of different biological activities, many of which are of considerable value to society. NPs or NP-inspired compounds represent ~65% of all small-molecule approved drugs 1 used in medicine to treat infectious diseases, cancers or as immunosuppressants 2 , and they are also applied extensively in agriculture 3. While NPs have been an extremely productive source of new leads for the chemicals that are integral to modern life, rapid increases in resistance to antibiotics, cancer chemotherapies and pesticides pose significant threats to medicine and agriculture 4,5 .

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“…Therefore, we sought to identify additional DKP dimerases through sequence similarity network (SSN) analysis. 23 Using NascB and NznB as in silico probes, along with known DKP functionalizing P450s from the thaxtomin (12), 24 pulcherrimic acid (13), 25 guanitrypmycin (14), [26][27][28][29] bicyclomycin (15), 30,31 and mycocyclosin (16) 32, 33 BGCs, we mined the NCBI database to identify candidate DKP dimerases. The resulting sequences were assembled into a SSN, which revealed a series of nodes clustered with NascB and NznB.…”

mentioning

confidence: 99%

Structure and Function of NzeB, a Versatile C–C and C–N Bond-Forming Diketopiperazine Dimerase

Shende¹,

Khatri²,

Newmister³

et al. 2020

J. Am. Chem. Soc.

108

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The dimeric diketopiperazine (DKPs) alkaloids are a diverse family of natural products (NPs) whose unique structural architectures and biological activities have inspired the development of new synthetic methodology to access these molecules. However, catalystcontrolled methods that enable the selective formation of constitutional and stereoisomeric dimers from a single monomer are lacking. To resolve this long-standing synthetic challenge, we sought to characterize the biosynthetic enzymes that assemble these NPs for application in biocatalytic syntheses. Genome mining enabled identification of the cytochrome P450, NzeB (derived from Streptomyces sp. NRRL F-5053), which catalyzes both intermolecular carbon-carbon (C-C) and carbon-nitrogen (C-N) bond formation, generating all currently known DKP dimer scaffolds isolated from bacterial sources. To identify the molecular basis for the flexible site-, stereo-, and chemoselectivity of NzeB, we obtained high-resolution crystal structures (1.5Å) of the protein in complex with native and non-native substrates. This, to our knowledge, represents the first crystal structure of an oxidase catalyzing direct, intermolecular C-H amination. Site-directed mutagenesis was employed to assess the role individual active site residues play in guiding selective DKP dimerization. Finally, computational approaches were employed to evaluate plausible mechanisms regarding NzeB function and its ability to catalyze both CC and C-N bond formation. These results provide a structural and computational rationale for the catalytic versatility of NzeB, as well as new insights into variables that control selectivity of CYP450 diketopiperazine dimerases. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website.

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

Cited by 24 publications

References 47 publications

Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes

Discovery and Biosynthesis of the Antibiotic Bicyclomycin in Distantly Related Bacterial Classes

The hidden enzymology of bacterial natural product biosynthesis

Structure and Function of NzeB, a Versatile C–C and C–N Bond-Forming Diketopiperazine Dimerase

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