Functional and Structural Studies on the Esperamicin Thioesterase and Progress toward Understanding Enediyne Core...

Area of Science:

• Natural Product Biosynthesis
• Structural Biology
• Enzyme Catalysis

Background:

• Enediynes are potent natural products with significant antitumor and antibacterial activities.
• Their biosynthesis involves a linear polyene precursor synthesized by iterative type I polyketide synthases (PKSEs).
• A crucial step is the release of this polyene precursor by a discrete thioesterase (TE).

Purpose of the Study:

• To elucidate the mechanism by which enediyne thioesterases (TEs) mediate polyene precursor production.
• To determine the structural basis for the interaction between PKSEs and TEs in enediyne biosynthesis.
• To identify key catalytic residues involved in the TE-mediated release of the polyene precursor.

Main Methods:

• X-ray crystallography was employed to solve the structure of wild-type and mutant enediyne TEs.
• Site-directed mutagenesis was used to probe the function of specific amino acid residues.
• Heterologous coexpression of PKSEs and TEs, along with in vitro activity assays, were performed.

Main Results:

• The crystal structure of wild-type EspE7 and an EspE7 mutant complexed with a fatty acyl-CoA ligand were determined.
• Structural and biochemical data strongly implicate Glu68 in EspE7 as a key catalytic residue.
• Combinations of PKSEs and TEs produced 1,3,5,7,9,11,13-pentadecaheptaene (1) as the major product, supporting its role as the sole precursor.

Conclusions:

• Enediyne TEs likely function via a hydrolysis mechanism, similar to Pseudomonas sp. 4-HB-CoA TE, with Glu68 being critical.
• The findings provide strong evidence for a conserved catalytic mechanism among enediyne TEs.
• 1,3,5,7,9,11,13-pentadecaheptaene (1) is confirmed as the universal precursor for all enediyne cores.