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

Abstract

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.

Bacterial Hydrazine Biosynthetic Pathways Featuring Cupin/Methionyl tRNA Synthetase‐like Enzymes

“…The latter enzyme is a homolog of diazotases, which are involved in nitrite-dependent NÀ N bond formation in the biosynthesis of cremeomycin, [10] tasikamide, [12] avenalumic acid, [13a] p-coumaric acid, [13b] and spinamycin. [14] The biosynthesis of triacsins begins with the NMO Tri26 and the hydrazine synthetase Tri28 to produce the precursor of the first NÀ N bond: Lys-Gly. This undergoes oxidative CÀ N bond cleavage by the FAD-dependent oxidase Tri27 to give HAA.…”

“…Their two consecutive N−N bonds are constructed by two distinct N−N bond‐forming mechanisms: one is produced at the early stage of biosynthesis by the hydrazine synthetase Tri28 as a form of Lys‐Gly, whereas the other is generated at the late stage of biosynthesis by the nitrite‐utilizing N ‐nitrosating enzyme Tri17. The latter enzyme is a homolog of diazotases, which are involved in nitrite‐dependent N−N bond formation in the biosynthesis of cremeomycin, [10] tasikamide, [12] avenalumic acid, [13a] p ‐coumaric acid, [13b] and spinamycin [14] …”

“…The major biosynthetic route of the diazo group is mediated by diazotases, which belong to the ANL superfamily usually activating carboxylic acids in an ATP‐dependent manner. Characterized diazotases include CreM, [10] Aha11, [12] AvaA6, [13a] SpiA7, [14] and CmaA6 [13b] . Diazotases activate nitrite with ATP and couple it with aryl‐amines, usually possessing electron‐donating hydroxy groups at ortho‐ or para ‐positions, to generate diazo groups.…”

Phylogeny‐guided Characterization of Bacterial Hydrazine Biosynthesis Mediated by Cupin/methionyl tRNA Synthetase‐like Enzymes

“…In addition to the well-recognized NÀ N bond-forming enzymes, such as nitrous reductase (NOR), [1] hydrazine synthase (HZS), [2] and P450nor, [3] which are all involved in energy production and denitrification, [4] recent studies revealed several distinct protein families that catalyze NÀ N bond-formation in bacterial secondary metabolism. [5][6][7][8] Representatives include diazotases such as CreM [9] and its homologs, [10][11][12][13] the piperazate synthase KtzT, [14] the N-nitrosating enzyme SznF/StzF, [15,16] two distinct membrane-bound proteins AzpL [17] and VlmO, [18] and didomain proteins with a metal-binding cupin domain and a methionyl-tRNA synthetase (MetRS)-like domain. [10,18a,19-22] The family of cupin/MetRS-like didomain proteins will be referred to as "hydrazine synthetases (HSs)" hereafter, as they utilize ATP to synthesize hydrazine.…”

Hot off the Press

“…The biosynthetic gene cluster of the antifungal compound, spinamycin 28 , a hydrazine-containing aryl polyene from Streptomyces albospinus JCM3399 has been characterised. 27 Feeding studies revealed that one of the nitrogen atoms of the hydrazide moiety is derived from nitrous acid, while in vitro analysis showed that an AMP-dependent ligase, SpiA7 generates a diazonium salt, which is postulated as a key intermediate for N–N bond formation. Feeding studies and NMR analysis have revealed the chemical structure of the organoarsenic metabolite, bisenarsan 29 , from the actinomycete, Streptomyces lividans 1326.…”