Making and Breaking Leupeptin Protease Inhibitors in Pathogenic Gammaproteobacteria

Leupeptin is a bacterial small molecule that is used worldwide as a protease inhibitor. However, its biosynthesis and genetic distribution remain unknown. We identified a family of leupeptins in gammaproteobacterial pathogens, including Photorhabdus, Xenorhabdus, and Klebsiella species, amongst others. Through genetic, metabolomic, and heterologous expression analyses, we established their construction by discretely expressed ligases and accessory enzymes. In Photorhabdus species, a hypothetical protein required for colonizing nematode hosts was established as a new class of proteases. This enzyme cleaved the tripeptide aldehyde protease inhibitors, leading to the formation of “pro‐pyrazinones” featuring a hetero‐tricyclic architecture. In Klebsiella oxytoca, the pathway was enriched in clinical isolates associated with respiratory tract infections. Thus, the bacterial production and proteolytic degradation of leupeptins can be associated with animal colonization phenotypes. Leupeptin, a broad‐spectrum protease inhibitor, is used worldwide in protein isolation and has been established as a chemical model in autophagy and immunoproteasome research. The leupeptin pathway was identified in gammaproteobacterial pathogens and associated with animal colonization phenotypes. A new type of protease transforms the leupeptins into novel heterotricyclic „pro‐pyrazinones.“