Presentation Information
[1MENP-10]Biosynthetic studies on the nucleoside antibiotic amipurimycin
○Taro Shiraishi1, Genki Hibi2, Tomohisa Kuzuyama2,3 (1. Department of Chemistry, Graduate School of Science, Kyoto University (Japan), 2. Graduate School of Agricultural and Life Sciences, The University of Tokyo (Japan), 3. Collaborative Research Institute For Innovative Microbiology, The University of Tokyo (Japan))
Keywords:
natural product,actinomycetes
[Purpose]
Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds feature a tripartite structure composed of a core saccharide, a nucleobase, and one or more amino acids. Amipurimycin, a PNA featuring an atypical nucleobase, 2-aminopurine, is particularly notable for its complex nine-carbon core saccharide and the noncanonical amino acid (–)-cispentacin. Despite its fascinating structure and properties, the biosynthetic mechanism of amipurimycin has heretofore eluded biochemical scrutiny. In this study, we aimed to identify its biosynthetic gene cluster (BGC) and elucidate the novel enzymatic reactions involved in its biosynthesis.
[Methods]
We utilized a comparative genomics approach combined with heterologous expression to identify the BGC. In addition, to elucidate the biosynthetic pathway of the unique amino acid (–)-cispentacin, we conducted in vivo feeding studies. Furthermore, we performed a series of in vitro reconstitution assays using purified recombinant enzymes to functionally characterize their specific biochemical roles. [Results]Through comparative genomics and successful heterologous expression, we identified the complete BGC for amipurimycin, designated as the amc cluster. Subsequent in vitro analysis of the cluster-encoded enzyme Amc18 established its crucial role as an ATP-grasp ligase responsible for the attachment of the pendant amino acid, (–)-cispentacin. Furthermore, comprehensive analysis of the amc cluster combined with feeding studies allowed us to propose a biosynthetic pathway for (–)-cispentacin. Finally, total in vitro reconstitution of these enzymes revealed the unprecedented biosynthetic machinery responsible for this noncanonical amino acid.
[Conclusion]
We successfully identified the amc cluster and elucidated the unprecedented biosynthetic pathway for the noncanonical amino acid (–)-cispentacin, including its final attachment via the ATP-grasp ligase Amc18. These findings provide profound biochemical insights into PNA biosynthesis and establish a vital foundation for the future rational engineering and creation of novel biomedical derivatives.
Peptidyl nucleoside antibiotics (PNAs) are a diverse class of natural products with promising biomedical activities. These compounds feature a tripartite structure composed of a core saccharide, a nucleobase, and one or more amino acids. Amipurimycin, a PNA featuring an atypical nucleobase, 2-aminopurine, is particularly notable for its complex nine-carbon core saccharide and the noncanonical amino acid (–)-cispentacin. Despite its fascinating structure and properties, the biosynthetic mechanism of amipurimycin has heretofore eluded biochemical scrutiny. In this study, we aimed to identify its biosynthetic gene cluster (BGC) and elucidate the novel enzymatic reactions involved in its biosynthesis.
[Methods]
We utilized a comparative genomics approach combined with heterologous expression to identify the BGC. In addition, to elucidate the biosynthetic pathway of the unique amino acid (–)-cispentacin, we conducted in vivo feeding studies. Furthermore, we performed a series of in vitro reconstitution assays using purified recombinant enzymes to functionally characterize their specific biochemical roles. [Results]Through comparative genomics and successful heterologous expression, we identified the complete BGC for amipurimycin, designated as the amc cluster. Subsequent in vitro analysis of the cluster-encoded enzyme Amc18 established its crucial role as an ATP-grasp ligase responsible for the attachment of the pendant amino acid, (–)-cispentacin. Furthermore, comprehensive analysis of the amc cluster combined with feeding studies allowed us to propose a biosynthetic pathway for (–)-cispentacin. Finally, total in vitro reconstitution of these enzymes revealed the unprecedented biosynthetic machinery responsible for this noncanonical amino acid.
[Conclusion]
We successfully identified the amc cluster and elucidated the unprecedented biosynthetic pathway for the noncanonical amino acid (–)-cispentacin, including its final attachment via the ATP-grasp ligase Amc18. These findings provide profound biochemical insights into PNA biosynthesis and establish a vital foundation for the future rational engineering and creation of novel biomedical derivatives.
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