Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation.

Area of Science:

• Biochemistry
• Synthetic Biology
• Drug Discovery

Background:

• Antibiotic resistance necessitates new antimicrobial drugs, particularly macrolides.
• Polyketide synthase (PKS) thioesterases (TEs) catalyze macrolactone formation, crucial for macrolide antibiotics.
• Limited understanding of TE mechanisms hinders biocatalyst development for diverse substrates.

Purpose of the Study:

• To elucidate the mechanism of TE substrate selectivity in macrolactone formation.
• To engineer TEs as biocatalysts for a wider range of natural and non-natural substrates.
• To understand how TEs direct nucleophilic attack for macrolactone ring closure.

Main Methods:

• Acyl-enzyme intermediates were trapped as stable amides by substituting the active site serine hydroxyl with 1,3-diaminopropionic acid (DAP).
• DAP-modified TEs (TE_DAP) from pikromycin and erythromycin pathways were purified.
• TE_DAP variants were tested with various polyketide intermediates, and crystal structures were determined.

Main Results:

• Erythromycin TE exhibited permissive substrate selectivity, while pikromycin TE was selective for its native substrates.
• Crystal structure of pikromycin TE_DAP revealed a curled heptaketide substrate with high shape complementarity to the active site.
• Distinct acyl cavity shapes were observed across different TEs, including those from juvenimicin, tylosin, and fluvirucin biosynthesis.

Conclusions:

• TEs exhibit varied substrate selectivity based on active site architecture, particularly the acyl cavity.
• Engineering TEs with DAP provides insights into substrate binding and catalytic mechanisms.
• Understanding TE structural diversity facilitates the engineering of novel biocatalysts for macrolide synthesis.