Drug precursor biosynthesis hinges on carrier-mediated ring formation

Nitrogen-nitrogen bond-containing cyclic compounds such as pyrazole, triazole, pyridazine and many others are critical building blocks for a variety of medicinal compounds, both natural and synthetic. The biosynthesis of some of these compounds hinges on the formation of nitrogen-nitrogen (N-N) single bonds between amino acids. However, the mechanisms by which a diversity of compounds is possible is poorly understood.

Dr. Kenichi Matsuda and Professor Toshiyuki Wakimoto at Hokkaido University led a team to elucidate the biosynthetic pathway of actinopyridazinone, an N-N bond-containing cyclic compound that is an important scaffold for synthetic drugs. Their findings were published in the journalAngewandte Chemie International Edition.

"Actinopyridazinone is produced byStreptomyces, a genus of bacteria that is the source of the majority of antibiotics of natural origin," Wakimoto explains. "It is the first natural compound known to possess a dihydropyridazinone ring. This ring is also known as a 'wonder nucleus,' as it has been extensively studied as a precursor for a wide range of drugs."

在以往的研究中,该团队使用生物信息学identify a group of gene sequences that are potentially involved in the biosynthesis of natural products that contain N-N bonds, and from these genome sequences, they discovered the novel class of compounds called actinopyradizones. With a series of genetic and biochemical experiments, they were also able to unveil the first steps in the pathway; in this study, they focused on understanding how the dihydropyridazone ring is formed.

The gene clusterapyis the biosynthetic gene cluster associated with actinopyradizone synthesis. It contains 17 potential genes; knockout studies indicated that ten of these --apy1,apy2,apy3,apy4,apy6,apy8,apy9, apy10, apy11andapy13-- were necessary for actinopyradizone synthesis. Biochemical analyses of the knockouts allowed the team to deduce that Apy3, an AMP-dependent synthetase/ligase, Apy4, a serine hydrolase, and Apy6, a carrier protein-rhodanese fusion, were the key proteins responsible for the formation of the dihydropyridazone ring.

"Apy6 functions as a carrier molecule; and Apy3 loads the intermediate compound onto Apy6," Matsuda elaborates. "Apy4 then catalyses the removal of an acetyl group (-COCH₃); the resulting molecule is unstable and spontaneously reacts to form a dihydropyridazone ring. The most notable feature of actinopyridazone biosynthesis is the unprecedented carrier protein-mediated machinery for dihydropyridazinone formation."

Matsuda said that this study is the first description of the biosynthetic pathway for actinopyradizone, and is only the second study to report the enzyme-dependent biosynthesis of a N-N bond-containing ring structure. The first such compound is piperazic acid, whose biosynthetic pathways are completely unrelated; hence, this study has also highlighted that the biosynthetic pathways of N-N bond-containing cyclic compounds are very diverse.