Presentation Information
[1MENP-03]A unified framework of nitrogen metabolism in THN-derived meroterpenoid biosynthesis
○Tomohiro Noguchi1,2, Tomohisa Kuzuyama1,2 (1. The University of Tokyo (Japan), 2. Collaborative Research Institute for Innovative Microbiology (Japan))
Keywords:
Natural Product Biosynthesis,Bacteria,PLP-dependent enzyme,Diazotization,Meroterpenoid
[Purpose]
THN-derived meroterpenoids such as furaquinocin and naphterpin are structurally diverse actinomycete natural products that share a conserved biosynthetic gene cassette, suggesting a common yet mechanistically unresolved pathway. However, how the amino-containing intermediate 8-amino-flaviolin (8-AF) is converted into final nitrogen-free products has remained unclear. We aimed to clarify the continuous nitrogen metabolic framework underlying THN-derived meroterpenoid biosynthesis.
[Method]
We combined gene inactivation and complementation, heterologous expression, in vitro enzymatic assays, isotope-labeling experiments, LC-MS/GC-MS analyses, and structural/computational studies including AlphaFold2-based modeling and molecular dynamics simulations.
[Results]
In the naphterpin pathway, NphE was identified as a pyridoxal 5′-phosphate-dependent enzyme that catalyzes an unprecedented oxidative transamination. NphE transfers an amino group from L-glutamate to mompain to generate 8-AF while reducing O2 to H2O2, and structural modeling supported an oxygen-accessible active-site cavity consistent with this bifunctional reactivity. Genetic and biochemical analyses established 8-AF as a genuine cryptic intermediate shared by THN-derived meroterpenoid pathways. In the furaquinocin pathway, Fur5 and Fur6 mediated reductive deamination of 8-AF through transient diazotization, replacing the amino group with hydrogen to yield the hydroquinone intermediate 1,2,4,5,7-pentahydroxynaphthalene. This reduced intermediate was required for downstream methylation, prenylation, and a SAM-independent intramolecular hydroalkoxylation catalyzed by the methyltransferase homolog Fur21, forming the characteristic oxygen-containing ring of furaquinocin.
[Consideration]
These results reveal a sequential nitrogen redox strategy in which oxidative transamination installs a cryptic amino group and reductive deamination subsequently removes it to direct scaffold remodeling and enable downstream biosynthetic transformations.
[Conclusion]
This study establishes a unified biosynthetic model for THN-derived meroterpenoids and uncovers two unusual enzymatic transformations linking a nitrogen-containing common intermediate to nitrogen-free final products. The findings expand the catalytic repertoire of PLP-dependent and diazotization-associated enzymes and provide mechanistic insights for enzyme evolution and natural product synthesis.
THN-derived meroterpenoids such as furaquinocin and naphterpin are structurally diverse actinomycete natural products that share a conserved biosynthetic gene cassette, suggesting a common yet mechanistically unresolved pathway. However, how the amino-containing intermediate 8-amino-flaviolin (8-AF) is converted into final nitrogen-free products has remained unclear. We aimed to clarify the continuous nitrogen metabolic framework underlying THN-derived meroterpenoid biosynthesis.
[Method]
We combined gene inactivation and complementation, heterologous expression, in vitro enzymatic assays, isotope-labeling experiments, LC-MS/GC-MS analyses, and structural/computational studies including AlphaFold2-based modeling and molecular dynamics simulations.
[Results]
In the naphterpin pathway, NphE was identified as a pyridoxal 5′-phosphate-dependent enzyme that catalyzes an unprecedented oxidative transamination. NphE transfers an amino group from L-glutamate to mompain to generate 8-AF while reducing O2 to H2O2, and structural modeling supported an oxygen-accessible active-site cavity consistent with this bifunctional reactivity. Genetic and biochemical analyses established 8-AF as a genuine cryptic intermediate shared by THN-derived meroterpenoid pathways. In the furaquinocin pathway, Fur5 and Fur6 mediated reductive deamination of 8-AF through transient diazotization, replacing the amino group with hydrogen to yield the hydroquinone intermediate 1,2,4,5,7-pentahydroxynaphthalene. This reduced intermediate was required for downstream methylation, prenylation, and a SAM-independent intramolecular hydroalkoxylation catalyzed by the methyltransferase homolog Fur21, forming the characteristic oxygen-containing ring of furaquinocin.
[Consideration]
These results reveal a sequential nitrogen redox strategy in which oxidative transamination installs a cryptic amino group and reductive deamination subsequently removes it to direct scaffold remodeling and enable downstream biosynthetic transformations.
[Conclusion]
This study establishes a unified biosynthetic model for THN-derived meroterpenoids and uncovers two unusual enzymatic transformations linking a nitrogen-containing common intermediate to nitrogen-free final products. The findings expand the catalytic repertoire of PLP-dependent and diazotization-associated enzymes and provide mechanistic insights for enzyme evolution and natural product synthesis.
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