Biosynthesis of Strained Amino Acids by a PLP-Dependent Enzyme through Cryptic Halogenation.

Amino acids (AAs) are modular building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides. While the 20 main proteinogenic AAs display relatively limited side chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis, but contain a broad array of structural features and functional groups. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase, PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack at C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans,  show that pazamine potentially inhibits ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Biosynthesis of Strained Amino Acids Through a PLP-Dependent Enzyme via Cryptic Halogenation.

Amino acids (AAs) are modular and modifiable building blocks which nature uses to synthesize both macromolecules, such as proteins, and small molecule natural products, such as alkaloids and non-ribosomal peptides (NRPs). While the 20 main proteinogenic AAs display relatively limited side-chain diversity, a wide range of non-canonical amino acids (ncAAs) exist that are not used by the ribosome for protein synthesis but contain a broad array of structural features and functional groups not found in proteinogenic AAs. In this communication, we report the discovery of the biosynthetic pathway for a new ncAA, pazamine, which contains a cyclopropane ring formed in two steps. In the first step, a chlorine is added onto the C4 position of lysine by a radical halogenase PazA. The cyclopropane ring is then formed in the next step by a pyridoxal-5'-phosphate-dependent enzyme, PazB, via an SN2-like attack onto C4 to eliminate chloride. Genetic studies of this pathway in the native host, Pseudomonas azotoformans, show that pazamine and its succinylated derivative, pazamide, potentially inhibit ethylene biosynthesis in growing plants based on alterations in the root phenotype of Arabidopsis thaliana seedlings. We further show that PazB can be utilized to make an alternative cyclobutane-containing AA. These discoveries may lead to advances in biocatalytic production of specialty chemicals and agricultural biotechnology.

Modular Chemoenzymatic Synthesis of GE81112 B1 and Related Analogues Enables Elucidation of Its Key Pharmacophores.

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.

Characterization of a Citrulline 4-Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine.

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.