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

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 (azs) cluster, which contains 21 genes, including those responsible for hydrazinoacetic acid (HAA) synthesis, was discovered using bioinformatics analysis of the Streptomyces fragilis genome. Azaserine was produced by the heterologous expression of the azs cluster in Streptomyces albus. In vitro 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 O-(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.