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Researchers identified and fully characterized the biosynthetic gene cluster responsible for producing sinefungin, a natural antimetabolite of S-adenosylmethionine, in Streptomyces incarnatus NRRL 8089. Contrary to decades-old hypotheses, the key carbon-carbon bond in sinefungin is formed by a vitamin B12-dependent radical SAM enzyme rather than a PLP-dependent mechanism, with the adenosyl group unusually derived from adenosylcobalamin through homolytic substitution. The pathway also employs a cryptic phosphorylation-dephosphorylation strategy and two specialized peptide aminoacyl-tRNA ligases (PEARLs) that attach alanine residues to the nucleoside scaffold using tRNA-activated amino acids, one of which can perform iterative elongation.
Why it matters
Understanding the complete biosynthetic logic of sinefungin opens avenues for engineered production of amino acid-nucleoside conjugates, a class of compounds with potential antimicrobial and antiviral applications. The discovery of an unusual adenosylcobalamin consumption mechanism and versatile PEARL enzymes also expands the known catalytic repertoire available for biocatalytic synthesis.
⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
Sinefungin is a potent nucleoside antimetabolite of S-adenosylmethionine (SAM), yet its biosynthesis has remained unclear for decades. Here we detail the identification and characterization of the complete sinefungin biosynthetic gene cluster (BGC) from Streptomyces incarnatus NRRL 8089. In vitro and in vivo analyses demonstrate that the defining carbon-carbon (C-C) bond is formed not by the long-hypothesized PLP-dependent process, but by a vitamin B12-dependent radical SAM enzyme. Using isotope-labeled cofactors and substrates, we provide evidence that the adenosyl group of sinefungin atypically originates from adenosylcobalamin via a homolytic SH2 substitution, establishing a rare instance where adenosylcobalamin is enzymatically consumed during the reaction. Furthermore, the pathway utilizes a cryptic phosphorylation-dephosphorylation strategy to control intermediate processing and substrate recognition. We also characterize two peptide aminoacyl-tRNA ligases (PEARLs) that append alanines onto the nucleoside scaffold using tRNA-activated amino acids. The PEARLs act directly on small molecules rather than macromolecular substrates, with one PEARL capable of iterative elongation. Finally, we leverage these enzymes in a reduced multi-enzyme cascade to biosynthesize sinefungin. Together, these findings redefine radical-mediated C-C bond formation and pearlin enzyme versatility, unlocking biocatalytic possibilities to produce amino acid-nucleoside conjugates.
Source: Unconventional biocatalytic strategies orchestrate synthesis of the nucleoside analog sinefungin