Identification of the Azaserine Biosynthetic Gene Cluster Implicates Hydrazine as an Intermediate to the Diazo Moiety

Abstract

Azaserine (1) is a natural product and nonproteinogenic amino acid containing a diazo group. Here we report the biosynthetic gene cluster for 1 from Glycomyces harbinensis. We then use isotopic feeding, gene deletion, and biochemical experiments to support a pathway whereby hydrazinoacetic acid (2) and a peptidyl carrier protein-loaded serine (3) are intermediates on route to the final natural product 1.

Conserved Enzymatic Cascade for Bacterial Azoxy Biosynthesis

“…These N–N moieties serve not only as important pharmacophores in a significant number of synthetic drugs but also as integral structural components in hundreds of bioactive natural products isolated from different sources (Figure a). , The potential utility of N–N forming enzymes in the realms of green chemistry and synthetic biology has sparked significant interest in understanding how N–N bonds are naturally constructed. − However, until very recently, dedicated N–N bond-forming enzymes are starting to be revealed. − For instance, the heme-dependent piperazate synthase was found to be responsible for catalyzing the hydrazine bond formation in piperazate, which is a commonly used building block employed in the assembly of many nonribosomal peptides . Moreover, a family of zinc-dependent cupin enzymes were found to provide hydrazine precursors to several N–N bond-containing metabolites with distinct chemical structures, including s56-p1, pyrazomycin, triacsins, and azaserine. − In addition, an iron-binding N -nitrosating enzyme , and a family of ATP-dependent diazotization enzymes were also identified. ,,, …”

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

“…[47] It is extensively used in the chemical biology field as an lglutamine mimic. [48] Biosynthetic gene clusters of azaserine were recently identified by three research groups independently, and three different designations were given: aza[alphabet], [23] aza[number], [25] and azs. [26] These contributions collectively elucidated most parts of azaserine biosynthesis: it largely overlaps with triacsin biosynthesis until the generation of the carrier protein-bound hydrazone unit HYAA, a key intermediate for hydrazone natural products.…”

“…The hydrazine products serve as the very first intermediates with NÀ N bonds and undergo various biosynthetic modifications to generate diverse NÀ N bond-containing functionalities, including hydrazone, [24] diazo, [23,25,26] and N-hydroxytriazene [11] groups, and NÀ N bond-containing heterocycles such as pyrazole [27][28][29][30] and dihydropyridazinone. [22] To date, hydrazine synthetases have been identified in the biosynthesis of six natural products, some with pharmaceutical relevance (Figure 1b).…”

Phylogeny‐guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase‐like Enzymes

“…Next, the Nterminal cupin domain catalyzes the NÀ N bond-forming rearrangement of the O-acyl hydroxylamine intermediate to give the amino acid-based hydrazine products. The resultant hydrazines are utilized as key precursors of NÀ N bondcontaining natural products, such as s56-p1 [19,24] (1; hydrazone), triacsins [10] (2; N-hydroxytriazene), azaserine [22,25,26] (3; diazo), formycins/pyrazomycin [27][28][29][30] (4, 5; pyrazole), and actinopyridazinones [21] (6, 7; dihydropyridazinone) (Figure 1b).…”