Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis

Area of Science:

• Natural Product Biosynthesis
• Enzyme Catalysis
• Organic Chemistry

Background:

• Anthraquinone-fused enediynes (AFEs) are cytotoxic molecules featuring a ten-membered enediyne core and an anthraquinone moiety.
• AFE cyclization involves F-ring epoxide formation, a site of significant structural diversity.
• Previous studies on tiancimycin A identified challenges in epoxide installation and lacked identified oxidases in its biosynthetic gene cluster.

Purpose of the Study:

• To identify the enzymes responsible for F-ring tailoring in tiancimycin biosynthesis.
• To elucidate the catalytic mechanisms of these novel enzymes.
• To expand the understanding of cofactorless oxygenase functions in natural product biosynthesis.

Main Methods:

• Comparative genomics to identify candidate enzymes within the tnm biosynthetic gene cluster.
• Biochemical characterization of purified enzymes.
• Structural analysis of enzyme-substrate interactions.

Main Results:

• Identification of two consecutively acting cofactorless oxygenases, TnmJ (methyltransferase fold) and TnmK2 (α/β-hydrolase fold).
• Demonstration that TnmJ and TnmK2 catalyze deformylation, epoxidation, and oxidative ring cleavage of the AFE F-ring.
• Evidence that the AFE anthraquinone moiety aids in catalyzing these reactions without exogenous cofactors.

Conclusions:

• TnmJ and TnmK2 are crucial for AFE F-ring tailoring in tiancimycin biosynthesis.
• These enzymes represent an underappreciated family of cofactorless oxygenases.
• The findings fill significant knowledge gaps in AFE biosynthesis and cofactorless enzyme mechanisms.