Seven enzymes create extraordinary molecular complexity in an uncultivated bacterium.
Uncultivated bacteria represent a massive resource of new enzymes and bioactive metabolites, but such bacteria remain functionally enigmatic. Polytheonamides are potent peptide cytotoxins produced by uncultivated bacteria that exist as symbionts in a marine sponge. Outside glycobiology, polytheonamides represent the most heavily post-translationally modified biomolecules that are derived from amino acids. The biosynthesis of polytheonamides involves up to 50 site-specific modifications to create a membrane-spanning β-helical structure. Here, we provide functional evidence that only seven enzymes are necessary for this process. They iteratively catalyse epimerization, methylation and hydroxylation of diverse amino acids. To reconstitute C-methylation, we employed the rarely used heterologous host Rhizobium leguminosarum to invoke the activities of two cobalamin-dependent C-methyltransferases. We observed 44 of the modifications to systematically unravel the biosynthesis of one of the most densely modified and metabolically obscure ribosome-derived molecules found in nature.
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.|Department of Biochemistry, Molecular Biology, and Biophysics and BioTechnology Institute, University of Minnesota-Twin Cities, St Paul, Minnesota 55108, USA.
Michael F
MF
Freeman
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Maximilian J
MJ
Helf
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Agneya
A
Bhushan
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Brandon I
BI
Morinaka
Institute of Microbiology, Eidgenössische Technische Hochschule (ETH) Zurich, Vladimir-Prelog-Weg 4, 8093 Zurich, Switzerland.
Jörn
J
Piel
D047908:Intracellular Signaling Peptides and Proteins; D008780:Methyltransferases / Q000378:metabolism*; D008968:Molecular Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D016961:Rhizobium leguminosarum / Q000201:enzymology* / Q000378:metabolism*
D047908:Intracellular Signaling Peptides and Proteins; D011506:Proteins; C111717:polytheonamide B; D008780:Methyltransferases