, ad iazo-containing amino acid, has been studied for more than 60 years as ap otent antitumor agent, but its biosynthesis has not been elucidated. Here we reveal the complete biosynthetic pathway of alazopeptin, the tripeptide Ala-DON-DON,w hichh as antitumor activity,bygene inactivation and in vitro analysis of recombinant enzymes.W ea lso established heterologous production of N-acetyl-DON in Streptomyces albus.D ON is synthesized from lysine by three enzymes and converted to alazopeptin by five enzymes and one carrier protein. Most interestingly,t ransmembrane protein AzpL was indicated to catalyze diazotization using 5-oxolysine and nitrous acid as substrates.Site-directed mutagenesis of AzpL indicated that the hydroxy group of Tyr-93 is important for the diazotization. These findings expand our knowledge of the enzymology of N À Nbond formation.

The diazo group is an important functional group that can confer biological activity to natural products owing to its high reactivity. Recent studies have revealed that diazo groups are synthesized from amino groups using nitrous acid in secondary metabolites of actinomycetes. However, genome database analysis indicated that there are still many diazo group‐biosynthesizing enzymes for unknown biosynthetic pathways. Here, we discovered an avenalumic acid biosynthesis gene cluster in Streptomyces sp. RI‐77 by genome mining of enzymes involved in diazo group formation. Through heterologous expression, the gene cluster was revealed to direct avenalumic acid (AVA) biosynthesis via 3‐aminoavenalumic acid (3‐AAA). In vitro enzyme assays showed that AvaA6 and AvaA7 catalyzed the diazotization of 3‐AAA using nitrous acid and substitution of the diazo group for hydride to synthesize AVA, respectively. This study revealed an unprecedented pathway for amino group removal via diazotization.

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α‐methyl‐l‐serine to synthesize a 4‐methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head‐to‐tail homodimeric structures of FmoA3 by X‐ray crystallography (apo‐form, with adenylyl‐imidodiphosphate and α‐methyl‐l‐seryl‐AMP) and cryogenic electron microscopy single particle analysis, and performed site‐directed mutagenesis experiments. The data revealed that α‐methyl‐l‐serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3’s Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α‐methyl‐l‐serine to synthesize a 4‐methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head‐to‐tail homodimeric structures of FmoA3 by X‐ray crystallography (apo‐form, with adenylyl‐imidodiphosphate and α‐methyl‐l‐seryl‐AMP) and cryogenic electron microscopy single particle analysis, and performed site‐directed mutagenesis experiments. The data revealed that α‐methyl‐l‐serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3’s Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.

Natural products containing nitrogen−nitrogen (N−N) bonds have attracted much attention because of their bioactivities and chemical features. Several recent studies have revealed the nitrous aciddependent N−N bond-forming machinery. However, the catalytic mechanisms of hydrazide synthesis using nitrous acid remain unknown. Herein, we focused on spinamycin, a hydrazide-containing aryl polyene produced by Streptomyces albospinus JCM3399. In the S. albospinus genome, we discovered a putative spinamycin biosynthetic gene (spi) cluster containing genes that encode a type II polyketide synthase and genes for the secondary metabolism-specific nitrous acid biosynthesis pathway. A gene inactivation experiment showed that this cluster was responsible for spinamycin biosynthesis. A feeding experiment using stable isotope-labeled sodium nitrite and analysis of nitrous acid-synthesizing enzymes in vitro strongly indicated that one of the nitrogen atoms of the hydrazide group was derived from nitrous acid. In vitro substrate specificity analysis of SpiA3, which is responsible for loading a starter substrate onto polyketide synthase, indicated that N−N bond formation occurs after starter substrate loading. In vitro analysis showed that the AMPdependent ligase SpiA7 catalyzes the diazotization of an amino group on a benzene ring without a hydroxy group, resulting in a highly reactive diazo intermediate, which may be the key step in hydrazide group formation. Therefore, we propose the overall biosynthetic pathway of spinamycin. This study expands our knowledge of N−N bond formation in microbial secondary metabolism.