In vitro characterization of nonribosomal peptide synthetase-dependent O-(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine

Abstract

Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway of azaserine. The putative azaserine biosynthetic gene (azs) cluster, which contains...

Conserved Enzymatic Cascade for Bacterial Azoxy Biosynthesis

“…To further probe the role of molecular oxygen in this process, we carried out VlmB-catalyzed reaction under anaerobic conditions and observed that the production of 3 was strictly dependent on the presence of molecular oxygen (Figure 3e). Moreover, LC-HR-MS analysis of the product 3 generated under an 18 O 2 atmosphere showed that the mass value for 3 increased by 2 Da (from m/z 191.1023 to 193.1066), demonstrating that the oxygen atom incorporated into the azoxy moiety derived from molecular oxygen (Figure 3f). Taken together, these results demonstrated that VlmB operates as a nonheme diiron azoxy synthase that catalyzes the reaction of 4 with O 2 to form 3.…”

“…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

“…Azs designation was employed here to maintain consistency throughout the azaserine biosynthetic pathway. *Different conclusions were obtained regarding the conversion of PCP‐tethered succinyl‐HAA to HYAA, a process involving either one enzyme (path a) [11,23] or two enzymes (path b) [26] . Steps without experimental validations are shown with dashed arrows.…”

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).…”