Covalent Linkage Mediates Communication between ACP and TE Domains in Modular Polyketide Synthases

Abstract

Polyketide natural products such as erythromycin A and epothilone are assembled on multienzyme polyketide synthases (PKSs), which consist of modular sets of protein domains. Within these type I systems, the fidelity of biosynthesis depends on the programmed interaction among the multiple domains within each module, centered around the acyl carrier protein (ACP). A detailed understanding of interdomain communication will therefore be vital for attempts to reprogram these pathways by genetic engineering. We report here that the interaction between a representative ACP domain and its downstream thioesterase (TE) is mediated largely by covalent tethering through a short "linker" region, with only a minor energetic contribution from protein-protein molecular recognition. This finding helps explain in part the empirical observation that TE domains can function out of their normal context in engineered assembly lines, and supports the view that overall PKS architecture may dictate at least a subset of interdomain interactions.

Systematic Domain Swaps of Iterative, Nonreducing Polyketide Synthases Provide a Mechanistic Understanding and Rationale For Catalytic Reprogramming

“…9,27 Acyl-ACP species have been shown to negotiate client domain interactions in modular PKSs and FASs. 42,43 Indeed, in some of the combinatorial reactions containing the CTB1 and ACAS PT domains, the noncognate PT influenced the final chain length, directing formation of a longer or shorter intermediate, respectively.…”

An Unusual Thioesterase Promotes Isochromanone Ring Formation in Ajudazol Biosynthesis

“…DEBSTEI and TylTEII were produced according to the published protocols. [17,33] All six recombinant proteins were then purified to homogeneity by affinity chromatography (Figure 3), and the identities of the newly engineered AjuTE, JerTE, DisTE, and SpiTEI were confirmed by MALDI-MS analysis.…”

“…The expression constructs for the "type I" TEs (Aju, Jer, Spi, and Dis) were designed to incorporate the majority of the linker between the TE and the upstream ACP domain (starting five amino acids C-terminal to the ACP), a strategy that had already proved successful for the expression of DEBSTEI. [33] The genes were cloned into vector pET28b + in order to obtain N-terminally His 6 -tagged proteins, and expression was carried out in E. coli Rosetta BL21(DE3)pLysS/RARE cells. DEBSTEI and TylTEII were produced according to the published protocols.…”

“…After induction, the cells were cultivated at 16 8C overnight, then harvested by centrifugation at 15 344 g. Expression of TylTEII and DEBSTEI was performed as described previously. [17,33] Purification of all proteins was carried out at 4 8C by using an ¾kta-Prime Purification System (GE Healthcare). Cell pellets were resuspended in buffer A (20 mm Tris, pH 7.8, 200 mm NaCl, 10 % glycerol, 10 mm imidazole; 20 mL).…”

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis

“…5). Transfer of the penultimate bicyclic intermediate 6 from the PT domain to the TE/CLC domain and its final cyclization is facilitated by the direct tethering (27) of the ACP and TE/CLC domains. The PPT-ACP phosphodiester linkage then mediates docking of the substrate-bound ACP domain to the TE/CLC active site.…”