Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase

Background: Polyketides are important compounds with antibiotic and antican cer activities. Several modular polyketide syntheses (PKSs) contain a termi nal thioesterase (TE) domain probably responsible for the release and conco mitant cyclization of the fully processed polyketide chain. Because the TE domain influences qualitative aspects of product formation by engineered PK Ss, its mechanism and specificity are of considerable interest. Results: The TE domain of the 6-deoxyerythronolide B synthase was overexpre ssed in Escherichia coli, When tested against a set of N-acetyl cysteamine thioesters the TE domain did not act as a cyclase, but showed significant h ydrolytic specificity towards substrates that mimic important features of i ts natural substrate. Also the overall rate of polyketide chain release was strongly enhanced by a covalent connection between the TE domain and the t erminal PKS module (by as much as 100-fold compared with separate TE and PK S 'domains'), Conclusions: The inability of the TE domain alone to catalyze cyclization s uggests that macrocycle formation results from the combined action of the T E domain and a PKS module, The chain-length and stereochemical preferences of the TE domain might be relevant in the design and engineered biosynthesi s of certain novel polyketides. Our results also suggest that the TE domain might loop back to catalyze the release of polyketide chains from both ter minal and pre-terminal modules, which may explain the ability of certain na turally occurring PKSs, such as the picromycin synthase, to generate both 1 2-membered and 14-membered macrolide antibiotics.