The GE81112 complex has garnered much interest due to its broad antimicrobial properties and unique ability to inhibit bacterial translation initiation. Herein we report the use of a chemoenzymatic strategy to complete the first total synthesis of GE81112 B1. By pairing iron and α-ketoglutarate dependent hydroxylases found in GE81112 biosynthesis with traditional synthetic methodology, we were able to access the natural product in 11 steps (longest linear sequence). Following this strategy, 10 GE81112 B1 analogues were synthesized, allowing for identification of its key pharmacophores. A key feature of our medicinal chemistry effort is the incorporation of additional biocatalytic hydroxylations in modular analogue synthesis to rapidly enable exploration of relevant chemical space.

The GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3‐hydroxy‐l‐pipecolic acid unit, little is known about the biosynthetic origins of the non‐proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4‐hydroxy‐l‐citrulline unit and establish the role of an iron‐ and α‐ketoglutarate‐dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4‐arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics‐guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.

We report a chemoenzymatic total synthesis of GE81112 B1 that employs biocatalytic hydroxylation to prepare two of the key monomers of the target natural product. By pairing this strategy with traditional organic chemistry, we were able to synthesize GE81112 B1 in 11 steps.

The GE81112 tetrapeptides are as mall family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3-hydroxy-l-pipecolic acid unit, little is known about the biosynthetic origins of the non-proteinogenic amino acid monomers of the natural product family.H ere,w e elucidate the biogenesis of the 4-hydroxy-l-citrulline unit and establish the role of an iron-and a-ketoglutarate-dependent enzyme (Fe/aKG) in the pathway.H omology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become as pecific 4-arginine hydroxylase.W es ubsequently demonstrate the utility of this engineered enzyme in the synthesis of adipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying abioinformatics-guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.

We functionally characterize a nonheme dioxygenase from GE81112 biosynthesis and identify it as a citrulline hydroxylase. A bioinformatics guided engineering was performed to alter the substrate specificity of the enzyme.