The first structure of a bacterial diterpene cyclase: CotB2

Area of Science:

• Biochemistry
• Structural Biology
• Natural Product Chemistry

Background:

• Sesquiterpenes and diterpenes are plant-derived secondary metabolites with valuable pharmaceutical properties.
• Their complex structures make microbial metabolic engineering a preferred production method over chemical synthesis.
• Bacterial diterpene cyclases are key enzymes in the biosynthesis of these compounds.

Purpose of the Study:

• To elucidate the structure and function of the bacterial diterpene cyclase CotB2.
• To explore the potential of CotB2 engineering for generating novel bioactive compounds.
• To establish a sustainable production route for a new antibiotic.

Main Methods:

• X-ray crystallography was used to determine the 1.64 Å resolution crystal structure of CotB2.
• Site-directed mutagenesis of active site residues in CotB2 was performed.
• The bioactivity of engineered products against bacterial strains, including multidrug-resistant Staphylococcus aureus, was assessed.

Main Results:

• The first crystal structure of bacterial diterpene cyclase CotB2 was determined.
• Mutagenesis of CotB2 yielded novel monocyclic, dicyclic, and tricyclic compounds with demonstrated bioactivity.
• A specific mutant, CotB2(W288G), produced a new antibiotic, (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, effective against multidrug-resistant Staphylococcus aureus.

Conclusions:

• The CotB2 structure provides insights into diterpene cyclase reaction mechanisms.
• Engineered CotB2 variants offer a pathway to new bioactive natural products.
• Sustainable industrial-scale production of a novel antibiotic against resistant bacteria is now feasible.