Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium

Freeman, Michael F.; Helf, Maximilian J.; Bhushan, Agneya; Morinaka, Brandon I.; Piel, Jörn

doi:10.1038/nchem.2666

Cited by 115 publications

(156 citation statements)

References 45 publications

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“…From these co-expression studies, 13 of the 17 expected C -methylations on the PoyA peptide were detected and localized. 26 It was gleaned that the C -methyltransferase activity was dependent on the PoyA core region with PoyC acting on residues 1–21 and PoyB on residues 23–49 (Figure 3). 26 In vitro experiments using a synthetic PoyA 1-15 peptide led to the formation of 8 (Figure 4) that contained only one methylation at L-Val 14 .…”

Section: Ripp Biosynthetic Reactions Catalyzed By Rsam Enzymesmentioning

confidence: 99%

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

2017

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Ribosomally synthesized and post-translationally modified peptides (RiPPs) display a diverse range of structures and continue to expand as a natural product class. Accordingly, RiPPs exhibit a wide array of bioactivities, such as broad and narrow spectrum growth suppressors, antidiabetics, as well as antinociception and anticancer agents. Owing to these properties, and the complex repertoire of post-translational modifications (PTMs) that give rise to these molecules, RiPP biosynthesis has been intensely studied. RiPP biosynthesis often involves enzymes that perform unique chemistry with intriguing reaction mechanisms, which attract chemists and biochemists alike to study and re-engineer these pathways. One particular type of RiPP biosynthetic enzyme is the so-called radical S-adenosylmethionine (rSAM) enzyme, which utilize radical-based chemistry to install several distinct PTMs. Here, we describe the rSAM enzymes characterized over the past decade that catalyze six reactions types from several RiPP biosynthetic pathways. We present the current state of mechanistic understanding and conclude with possible directions for future characterization of this enzyme family.

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Section: Ripp Biosynthetic Reactions Catalyzed By Rsam Enzymesmentioning

confidence: 99%

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

2017

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“…5,6 Very recently, the B 12 /radical SAM mediated conversion of isopropyl glycine to t -butyl glycine in the polytheoamide propeptide was reported (Figure 1D). 7,8 The latter two enzymes are the only t -butyl biosynthesis enzymes that have been experimentally reconstituted.…”

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confidence: 99%

Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme

et al. 2017

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The biosynthesis of branched alkoxy groups, such as the unique t-butyl group found in a variety of natural products, is still poorly understood. Recently, cystobactamids were isolated and identified from Cystobacter sp as novel antibacterials. These metabolites contain an isopropyl group proposed to be formed using CysS, a cobalamin-dependent radical S-adenosylmethionine (SAM) methyltransferase. Here, we reconstitute the CysS-catalyzed reaction, on p-aminobenzoate thioester substrates, and demonstrate that it not only catalyzes sequential methylations of a methyl group to form ethyl and isopropyl groups but remarkably also sec-butyl and t-butyl groups. To our knowledge, this is the first in vitro reconstitution of a cobalamin-dependent radical SAM enzyme catalyzing the conversion of a methyl group to a t-butyl group.

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“…By metagenomic, single-particle genomic, and functional studies, we and collaborators recently provided evidence that almost all known bioactive polyketides and modified peptides previously reported from a Japanese chemotype of T. swinhoei (termed "chemotype Y") are produced by a single member of the complex microbiome named "Candidatus Entotheonella factor" TSY1 (15)(16)(17)(18). In this sponge, the bacterium co-occurs with a second Ca.…”

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confidence: 99%

Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges

Lackner

Peters

Helfrich

et al. 2017

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

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126

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The as-yet uncultured filamentous bacteria "Candidatus Entotheonella factor" and "Candidatus Entotheonella gemina" live associated with the marine sponge Theonella swinhoei Y, the source of numerous unusual bioactive natural products. Belonging to the proposed candidate phylum "Tectomicrobia," Candidatus Entotheonella members are only distantly related to any cultivated organism. The Ca. E. factor has been identified as the source of almost all polyketide and modified peptides families reported from the sponge host, and both Ca. Entotheonella phylotypes contain numerous additional genes for as-yet unknown metabolites. Here, we provide insights into the biology of these remarkable bacteria using genomic, (meta)proteomic, and chemical methods. The data suggest a metabolic model of Ca. Entotheonella as facultative anaerobic, organotrophic organisms with the ability to use methanol as an energy source. The symbionts appear to be auxotrophic for some vitamins, but have the potential to produce most amino acids as well as rare cofactors like coenzyme F 420 . The latter likely accounts for the strong autofluorescence of Ca. Entotheonella filaments. A large expansion of protein families involved in regulation and conversion of organic molecules indicates roles in host-bacterial interaction. In addition, a massive overrepresentation of members of the luciferase-like monooxygenase superfamily points toward an important role of these proteins in Ca. Entotheonella. Furthermore, we performed mass spectrometric imaging combined with fluorescence in situ hybridization to localize Ca. Entotheonella and some of the bioactive natural products in the sponge tissue. These metabolic insights into a new candidate phylum offer hints on the targeted cultivation of the chemically most prolific microorganisms known from microbial dark matter.

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Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium

Cited by 115 publications

References 45 publications

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

Radical S-Adenosylmethionine Enzymes Involved in RiPP Biosynthesis

Biosynthesis of Branched Alkoxy Groups: Iterative Methyl Group Alkylation by a Cobalamin-Dependent Radical SAM Enzyme

Insights into the lifestyle of uncultured bacterial natural product factories associated with marine sponges

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