A thioesterase from an iterative fungal polyketide synthase shows macrocyclization and cross coupling activity and may play a role in controlling iterative cycling through product offloading

Abstract

Zearalenone, a fungal macrocyclic polyketide, is a member of the resorcylic acid lactone family. Herein, we characterize in vitro the thioesterase from PKS13 in zearalenone biosynthesis (Zea TE). The excised Zea TE catalyzes macrocyclization of a linear thioester-activated model of zearalenone. Zea TE also catalyzes the cross coupling of a benzoyl thioester with alcohols and amines. Kinetic characterization of the cross coupling is consistent with a ping-pong bi-bi mechanism, confirming an acyl-enzyme intermediate. Finally, the substrate specificity of the Zea TE indicates the TE may help control iterative cycling on PKS13 by rapidly offloading the final resorcylate-containing product.

Figure 1

The biosynthesis of zearalenone, an archetypical RAL.

Figure 2

Zea TE catalyzes macrocyclization of 14-member rings. A: HPLC analysis of authentic standards. B: HPLC analysis of a 30 min incubation of 15 µM Zea TE with 5 mM 6 in 50 mM phosphate buffer at pH 7.4, 23 °C. Negative controls lacking Zea TE showed no detectable macrocyclization activity.

Figure 3

HPLC analysis of Zea TE catalyzed cross-coupling reactions. A: Products and standards. B: Cross-coupling reaction with aniline. C: Cross-coupling reaction with butanol. Enzymatic reactions were incubated 3h with 5 µM Zea TE, 10 mM 9, 20 mM analine or butanol in 50 mM phosphate buffer at pH 7.4, 23 °C. Negative controls lacking Zea TE showed no detectable cross coupling products.