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Angewandte Chemie (International Ed. in... Jul 2023Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the...
Azaserine is a bacterial metabolite containing a biologically unusual and synthetically enabling α-diazoester functional group. Herein, we report the discovery of the azaserine (aza) biosynthetic gene cluster from Glycomyces harbinensis. Discovery of related gene clusters reveals previously unappreciated azaserine producers, and heterologous expression of the aza gene cluster confirms its role in azaserine assembly. Notably, this gene cluster encodes homologues of hydrazonoacetic acid (HYAA)-producing enzymes, implicating HYAA in α-diazoester biosynthesis. Isotope feeding and biochemical experiments support this hypothesis. These discoveries indicate that a 2-electron oxidation of a hydrazonoacetyl intermediate is required for α-diazoester formation, constituting a distinct logic for diazo biosynthesis. Uncovering this biological route for α-diazoester synthesis now enables the production of a highly versatile carbene precursor in cells, facilitating approaches for engineering complete carbene-mediated biosynthetic transformations in vivo.
Topics: Azaserine; Biosynthetic Pathways; Methane; Oxidation-Reduction; Multigene Family
PubMed: 37151182
DOI: 10.1002/anie.202304646 -
Chemical Science Aug 2023Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative...
characterization of nonribosomal peptide synthetase-dependent -(2-hydrazineylideneacetyl)serine synthesis indicates a stepwise oxidation strategy to generate the α-diazo ester moiety of azaserine.
Azaserine, a natural product containing a diazo group, exhibits anticancer activity. In this study, we investigated the biosynthetic pathway to azaserine. The putative azaserine biosynthetic gene () cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the genome. Azaserine was produced by the heterologous expression of the cluster in . enzyme assays using recombinant Azs proteins revealed the azaserine biosynthetic pathway as follows. AzsSPTF and carrier protein (CP) AzsQ are used to synthesize the 2-hydrazineylideneacetyl (HDA) moiety attached to AzsQ from HAA. AzsD transfers the HDA moiety to the C-terminal CP domain of AzsN. The heterocyclization (Cy) domain of the nonribosomal peptide synthetase AzsO synthesizes -(2-hydrazineylideneacetyl)serine (HDA-Ser) attached to its CP domain from l-serine and HDA moiety-attached AzsN. The thioesterase AzsB hydrolyzes it to yield HDA-Ser, which appears to be converted to azaserine by oxidation. Bioinformatics analysis of the Cy domain of AzsO showed that it has a conserved DxxxxD motif; however, two conserved amino acid residues (Thr and Asp) important for heterocyclization are substituted for Asn. Site-directed mutagenesis of two Asp residues in the DxxxxD motif (D193 and D198) and two substituted Asn residues (N414 and N447) indicated that these four residues are important for ester bond synthesis. These results showed that the diazo ester of azasrine is synthesized by the stepwise oxidation of the HAA moiety and provided another strategy to biosynthesize the diazo group.
PubMed: 37621439
DOI: 10.1039/d3sc01906c