Cyclization modes in type III polyketide synthases and the synthetic compounds used as probes for mechanistic studies.

Abstract

Type III polyketide synthases (PKS) catalyze different cyclization reactions of linear polyketide intermediates to produce polyketides with distinct ring structures. The mechanisms of pyrone and resorcinol ring formation in type III PKSs remain unknown, despite many years of research on this family of enzymes. Compounds, thought to be intermediates in these enzyme reactions, have been synthesized and used to probe the enzyme mechanisms. To study the mechanism of resorcinol ring formation catalyzed by STS and ArsB, triketo acids and 9-phenyl-3,5,7-trioxo-8-nonenoic and 3,5,7-trioxoeicosanoic (16a and 16b) and their structural analogs were synthesized. STS failed to produce any cyclized products from the synthetic compounds. ArsB also failed to convert 3,5,7-trioxoeicosanoic acid 16b to 5-tridecylresorcinol 14b. Instead, ArsB facilitated the production of 6-tridecylresorcylic acid 11b from the synthetic 16b and hindered the decarboxylation of 11b to 14b. These effects were also observed with ArsC, another acyl-CoA utilizing PKS that does not catalyze the resorcinol ring formation. Furthermore, the triketo acid 16b and its analogs inhibited the ArsB activity. These results indicate that neither 16b nor 11b is an intermediate and that aldol cyclization of the linear tetraketide thioester intermediate (15) precedes the hydrolysis of 15 in the ArsB catalyzed resorcinol ring formation. 3,5-Dioxooctanoic acid 32a was also synthesized in order to probe the lactonization mechanism in type III PKSs such as PpASCL and AthPKSA. It has been suggested that diketo acids are synthesized by these enzymes, released, and then lactonized in solution. The results of this study show that even prolonged incubation of 32a in buffer failed to result in any pyrone formation. Also, the enzymes were unable to convert the 32a to 4-hydroxy-6-tridecyl- 2-pyrone 33a, which led to the conclusion that pyrone formation is most likely an enzymatic process, occurring via direct lactonization of a CoA thioester.