A Natural Dihydropyridazinone Scaffold Generated from a Unique Substrate for a Hydrazine-Forming Enzyme

Abstract

Nitrogen−nitrogen bond-containing functional groups are rare, but they are found in a considerably wide class of natural products. Recent clarifications of the biosynthetic routes for such functional groups shed light onto overlooked biosynthetic genes distributed across the bacterial kingdom, highlighting the presence of yet-to-be identified natural products with peculiar functional groups. Here, the genome-mining approach targeting a unique hydrazine-forming gene led to the discovery of actinopyridazinones A (1) and B (2), the first natural products with dihydropyridazinone rings. The structure of actinopyridazinone A was unambiguously established by total synthesis. Biosynthetic studies unveiled the structural diversity of natural hydrazines derived from this family of N−N bond-forming enzymes.

Automated genome mining predicts combinatorial diversity and taxonomic distribution of peptide metallophore structures

“…Instead, the acyl-hydroxyornithine epimerase 29 VbsL is used to detect vicibactin. In two non-metallophore pathways, 30,31 hydroxyornithine or hydroxylysine are the substrates for N-N bond-forming enzymes. No clear bitscore separation could be achieved to eliminate these false positives, so two negative constraints were added: the presence of KtzT associated with biosynthesis of piperazates, 30 and MetRS-like , associated with several hydrazines.…”

“…No clear bitscore separation could be achieved to eliminate these false positives, so two negative constraints were added: the presence of KtzT associated with biosynthesis of piperazates, 30 and MetRS-like , associated with several hydrazines. 31 Unfortunately, false positives may still arise if the ornithine monooxygenase and the negative constraint genes are (distantly) located within the same BGC region (>30 kbp), such as in the himastatin locus (MIBiG BGC0001117).…”

Identification and Analysis of the Biosynthetic Gene Cluster for the Hydrazide-Containing Aryl Polyene Spinamycin

“…To examine the activity of SpiA7 in vitro, we used 3-aminocinnamic acid (3-ACA, 12) as an analogue of 4 because 4 was not available owing to the difficulty of chemical synthesis. When SpiA7 and 12 were incubated with ATP and MgCl 2 , the production of cinnamic acid (14) was observed by LC−MS analysis (Figure 4B). Since 3-diazocinnamic acid (3-DCA, 13), which was assumed to be the actual product of this in vitro enzyme reaction, was likely to be unstable, the diazo group of 13 seemed to be nonenzymatically decomposed to yield 14 (Figure 4A).…”

“…The azoxy moiety of azoxymycin is derived from the nonenzymatic coupling of two aromatic nitroso groups synthesized by the diiron oxygenase AzoC. , In addition, a heme-dependent enzyme (KtzT) synthesizes piperazic acid from N- hydroxyornithine . Spb40 homologues generate a hydrazine moiety by condensing an N -hydroxyamino acid with another amino acid. − Furthermore, the biosynthesis of several N–N bond-containing natural products produced by other microorganisms has also been studied. However, how the N–N bond is synthesized remains unclear. − …”

New azodyrecins identified by a genome mining-directed reactivity-based screening

“…A combination of reactivity-based screening and genome-based prioritization would allow the prediction of the producer of natural products with the functional groups of interest, leading to a higher rate of hits. We recently exploited genome mining targeting hydrazine synthetase and the generation of inorganic hydrazine N 2 H 4 in acid hydrolysate as an indicator of N–N bond-containing functional groups, which led to the discovery of actinopyridazinones with a unique dihydropyridazinone scaffold [ 11 ]. N 2 H 4 generates similarly in the acid hydrolysate of azoxy compounds, and this feature has been exploited as proof for the presence of N–N bonds in the plant-derived methyl azoxy compound macrozamin [ 4 ].…”