Identification of the Polyketide Biosynthetic Machinery for the Indolizidine Alkaloid Cyclizidine

Huang, Wei; Kim, Seong Jong; Liu, Joyce F.; Zhang, Wenjun

doi:10.1021/acs.orglett.5b02707

“…This reductive release mechanism is consistent with the identified activity of the R domain in Rv0101, which contains a conserved Ser/ Tyr/Lys catalytic triad and was demonstrated to catalyze a fourelectron thioester reduction using the purified truncated R domain and a synthetic substrate (17). We propose that the extra acetyl moiety found in INLP 1 is due to the activity of a promiscuous acetyltransferase endogenous to E. coli for possible detoxification, and similar acetylation events have been observed in other shunt product biosynthesis (26). An analogous biosynthetic pathway involving MmaA-E has also been proposed, differing in the chain length of the fatty acyl moiety (Fig.…”

Section: Proposed Biosynthetic Pathways For Inlps By the Five Conservedsupporting

confidence: 84%

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Harris

¹

,

Sato

²

,

Herman

³

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.biosynthetic enzymes | mycobacteria | metal transport S mall-molecule secondary metabolites are produced by microbes as chemical weapons to combat competing organisms or as communication signals to control complex processes such as virulence, morphological differentiation, biofilm formation, and metal acquisition (1-3). One of the most important classes of secondary metabolites are nonribosomal peptides, which are typically biosynthesized by modular nonribosomal peptide synthetases (NRPSs) in an assembly-line manner (4). Two NRPS-encoding gene clusters (mbt and Rv0096-0101) have been identified from the genome of Mycobacterium tuberculosis, the leading causative agent of tuberculosis that currently infects one-third of the world's population. Although the cluster of mbt has been characterized to biosynthesize mycobactin siderophores that form mycobactin-Fe(III) complexes for iron sequestration (5), the role of Rv0096-0101 remains obscure despite various biological studies that have indicated the production of a virulence factor by this gene cluster (6-14). For example, using transposon-site hybridization, Rv0098 to Rv0101 were predicted to be required for M. tuberculosis survival in a mouse model of infection (10). Consistently, a transposon insertion of Rv0097 attenuated M. tuberculosis growth and survival in mice (7).An in silico homology search has revealed that gene clusters homologous to Rv0096-0101 are conserved in pathogenic mycobacteria, such as M. bovis, M. leprae, M. marinum, M. ulcerans, and M. abscessus (Fig. 1), but absent in nonpathogenic mycobacteria such as M. smegmatis, providing further indication of the virulenceassociated nature of the locus product in mycobacteria. Interestingly, in addition to the genus of Mycobacterium, related operons are found in the phylum of Actinobacteria across genera including Streptomyces, Kutzneria, Nocardia, and Rhodococcus (Fig. 1), suggesting a widespread presence of this cluster. Further bioinformatic analysis has shown that five genes (Rv0097-0101) are conserved across all identified gene cluste...

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“…This reductive release mechanism is consistent with the identified activity of the R domain in Rv0101, which contains a conserved Ser/Tyr/Lys catalytic triad and was demonstrated to catalyze a four-electron thioester reduction using the purified truncated R domain and a synthetic substrate (17). We propose that the extra acetyl moiety found in 1 is due to the activity of a promiscuous acetyltransferase endogenous to E. coli for possible detoxification, and similar acetylation events have been observed in other shunt product biosynthesis (25). An analogous biosynthetic pathway involving MmaA-E has also been proposed, differing in the chain length of the fatty acyl moiety ( Fig.…”

Section: Proposed Biosynthetic Pathways For Inlps By the Five Conservsupporting

confidence: 81%

Biosynthesis of Isonitrile Lipopeptides by Conserved Non-ribosomal Peptide Synthetase Gene Clusters inActinobacteria

Harris

¹

,

Sato

²

,

Herman

³

et al. 2017

Preprint

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A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in intro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a new family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a non-heme iron(II)-dependent oxidase homologue, and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a non-ribosomal peptide synthetase (NRPS). In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.Significance StatementMycobacterium tuberculosis is the leading causative agent of tuberculosis (TB), of which millions of deaths occur annually. A putative lipopeptide biosynthetic gene cluster has been shown to be essential for the survival of this pathogen in hosts, and homologous gene clusters have also been found in all pathogenic mycobacteria and other species of Actinobacteria. We have identified the function of these gene clusters in making a new family of isonitrile lipopeptides. The biosynthesis has several unique features, including an unprecedented mechanism for isonitrile synthesis. Our results have further suggested that these biosynthetic gene clusters play a role in metal transport, and thus have shed light on a new metal transport system that is crucial for virulence of pathogenic mycobacteria.

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“…30 Gene clusters were putatively assigned to alkaloid biosynthesis by matching the length of the polyketide chain encoded in the BGC to corresponding compound carbon skeletons in combination with logical tailoring enzymes. In this fashion, BGCs were putatively assigned to cyclizidines, 100–102, 109 nigrifactin, 92, 93 streptazolin, streptazone E, 110 pyrindicin, 95–97 and latumcidin. Some of these NPs had received supporting biosynthetic characterization, such as cyclizidine, 31 while others with consistent biosynthetic logic, such as argimycins PI—IX, were discovered later.…”

Section: Amine-containing Natural Productsmentioning

confidence: 99%

Natural products from thioester reductase containing biosynthetic pathways

Mullowney

¹

,

McClure

²

,

Robey

³

et al. 2018

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Thioester reductase domains catalyze two- and four-electron reductions to release natural products following assembly on nonribosomal peptide synthetases, polyketide synthases, and their hybrid biosynthetic complexes. This reductive off-loading of a natural product yields an aldehyde or alcohol, can initiate the formation of a macrocyclic imine, and contributes to important intermediates in a variety of biosyntheses, including those for polyketide alkaloids and pyrrolobenzodiazepines. Compounds that arise from reductase-terminated biosynthetic gene clusters are often reactive and exhibit biological activity. Biomedically important examples include the cancer therapeutic Yondelis (ecteinascidin 743), peptide aldehydes that inspired the first therapeutic proteasome inhibitor bortezomib, and numerous synthetic derivatives and antibody drug conjugates of the pyrrolobenzodiazepines. Recent advances in microbial genomics, metabolomics, bioinformatics, and reactivity-based labeling have facilitated the detection of these compounds for targeted isolation. Herein, we summarize known natural products arising from this important category, highlighting their occurrence in Nature, biosyntheses, biological activities, and the technologies used for their detection and identification. Additionally, we review publicly available genomic data to highlight the remaining potential for novel reductively tailored compounds and drug leads from microorganisms. This thorough retrospecive highlights various molecularfamilies with especially privileged bioactivity while illuminating challenges and prospects toward accelerating the discovery of new, high value natural products.

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Identification of the Polyketide Biosynthetic Machinery for the Indolizidine Alkaloid Cyclizidine

Cited by 34 publications

References 33 publications

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria

Biosynthesis of Isonitrile Lipopeptides by Conserved Non-ribosomal Peptide Synthetase Gene Clusters inActinobacteria

Natural products from thioester reductase containing biosynthetic pathways

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