Carrier Protein Mediated Formation of the Dihydropyridazinone Ring in Actinopyridazinone Biosynthesis

Abstract

Heterocycles with nitrogen‐nitrogen (N−N) bonds are privileged building blocks of synthetic drugs. They are also found in natural products, although the biosynthetic logic behind them is poorly understood. Actinopyridazinones produced by Streptomyces sp. MSD090630SC‐05 possess unique dihydropyridazinone rings that have been studied as core nuclei in several approved synthetic therapeutics. Herein, we performed gene knockouts and in vitro biochemical experiments to elucidate the major steps in actinopyridazinone biosynthesis, including the unprecedented carrier protein mediated machinery for dihydropyridazinone formation.

Discovery of a Bacterial Hydrazine Transferase That Constructs the N-Aminolactam Pharmacophore in Albofungin Biosynthesis

“…We reasoned that putative FAD-dependent oxidoreductase Afn7 could be responsible for this step, catalyzing a C–N bond cleavage reaction utilizing 12 (Figure S15). Homologues of Afn7 have been shown to catalyze analogous transformations following the MetRS/cupin-catalyzed reactions in several other pathways. ,, Indeed, we observed that strain Δ afn7 produced approximately 70% less 1 relative to the parental strain and accumulated 5 , indicating the incomplete conversion of 5 to the final product (Figure c). All our attempts to express soluble Afn7 were fruitless.…”

Bacterial Hydrazine Biosynthetic Pathways Featuring Cupin/Methionyl tRNA Synthetase‐like Enzymes

“…zinone biosynthesis, the flavin-dependent oxidase Apy10 oxidizes the CÀ N bond on the other side of the NÀ N bond in the hydrazine intermediate DABA-Ala to give l-2-amino-4hydrazineylbutanoic acid (l-AHBA), which is the core precursor of the dihydropyridazinone scaffold. [36] These observations collectively indicate that either the hydroxylamine or amino acid parts of hydrazines can be integrated into the final products, and the site-selective CÀ N bond cleavage step is the major pathway-branching point that dictates their shapes. The released aldehydes (e. g., AASA from Lys-Gly, Lys-Glu) or α-keto acids (e. g., pyruvate from DABA-Ala) are the direct precursors of the corresponding amino acids and would be recycled by primary metabolism to serve as nitrogen carriers in hydrazine biosynthesis.…”

“…[22] Biochemical experiments have almost entirely elucidated the biosynthetic pathway of actinopyridazinone (apy), including the unique heterocyclization mechanism employing a distinctive carrier protein (Figure 7). [36] Similar to spb, tri, and aza, the hydrazine intermediate DABA-Ala undergoes oxidative CÀ N bond cleavage mediated by the FADdependent oxidase Apy10 to yield l-AHBA. Importantly, the regioselectivity of the CÀ N bond oxidation differs from that in HAA biosynthesis, leaving the NÀ N bond intact in the side chain of l-DABA.…”