Complete reconstitution of a highly reducing iterative polyketide synthase

Abstract

Highly reducing iterative polyketide synthases are large, multifunctional enzymes that make important metabolites in fungi, such as lovastatin, a cholesterol-lowering drug from Aspergillus terreus. We report efficient expression of the lovastatin nonaketide synthase (LovB) from an engineered strain of Saccharomyces cerevisiae, as well as complete reconstitution of its catalytic function in the presence and absence of cofactors (the reduced form of nicotinamide adenine dinucleotide phosphate and S-adenosylmethionine) and its partner enzyme, the enoyl reductase LovC. Our results demonstrate that LovB retains correct intermediates until completion of synthesis of dihydromonacolin L, but off-loads incorrectly processed compounds as pyrones or hydrolytic products. Experiments replacing LovC with analogous MlcG from compactin biosynthesis demonstrate a gate-keeping function for this partner enzyme. This study represents a key step in the understanding of the functions and structures of this family of enzymes.

Fig. 1

Proposed mechanism of dihydromonacolin L 1 synthesis by LovB and the accessory ER LovC. LovB (335 kDa) consists of eight discrete domains and operates iteratively to condense nine malonylCoA equivalents to yield the nonaketide product 1. Loading of the megasynthase by malonyl-CoA is presumably followed by decarboxylation to yield the acetyl starter unit (not shown). Each round of Claisen-condensation is catalyzed by the KS domain, while the growing polyketide is tethered to the phosphopantetheinyl (shown in squiggle line) arm of the ACP. After each condensation, the polyketide is subjected to a different combination of tailoring, which can include α-methylation by the MT domain, β-ketoreduction by the KR domain, β-dehydration by the DH domain, and α-β-enoylreduction by the dissociated LovC. The different tailoring permutations after each round of chain extension yield a triene-containing hexaketide thioester that can undergo a stereospecific Diels-Alder cyclization to yield the decalin portion of 1. After formation of the nonaketide, the chain is released to yield the ring open form 1. The acid form of 1 can undergo lactonization to yield 2.

Fig. 2

Polyketides synthesized by LovB under different assay conditions in the absence of LovC. (A) Trace i: reaction with LovB afforded the triketide lactone 3 which confirms the function of the minimal PKS domains of LovB; Trace ii: reaction with LovB and NADPH (2 mM) afforded a number of highly conjugated compounds 4-8; and Trace iii: reaction with LovB, NADPH (2 mM) and SAM (2 mM) afford the methylated, conjugated pyrones 9 and 10. All reactions used MatB to regenerate malonylCoA, and were extracted with ethyl acetate (EA)/acetic acid (AcOH) (99/1); (B) The fate of the tetraketide intermediate in the absence of SAM and LovC. The correct tailoring yields the key intermediate 11. In the absence of α-methylation, pyrones 4-6 can form readily through cyclization and release while the ketones 7-8 can form via hydrolysis and decarboxylation.

Fig. 3

Synthesis of 2 and 12 by LovB and dissociated ER in vitro. (A) LC-MS analysis of the synthesis 2 using purified LovB and LovC. Typical reaction contains 25 μM of LovB and LovC, 2 mM NADPH, 2 mM SAM, and the MatB malonyl-CoA regeneration components (See SI for details). The traces shown are the selected ion monitoring of desired ions in the positive ionization mode. Trace i: standard of 2 purified from A. nidulans (8). Trace ii: products recovered from the reaction following extraction with EA/AcOH (99/1). Trace iii: the reaction mixture was first treated with 1 M KOH at 65°C for 10 minutes to hydrolyze all attached polyketides, followed by acidification with 1 N HCl to pH 2.0, followed by extraction. Emergence of 2 indicates the products are not released by LovB under reaction conditions; Trace iv: products recovered from base-treated sample that used [2-¹³C]-malonate as the precursor. The +9 mass shift confirms the nonaketide backbone of 2 synthesized by LovB; Trace v: release of 2 from LovB by heterologous fungal G. zeae PKS13 TE (25 μM). No base hydrolysis is required to observed product release; (B) Synthesis of 12 in the absence of SAM requires a different ER (MlcG) from the compactin biosynthetic pathway. All reactions contain LovB, PKS13 TE, ER, and 2 mM NADPH, and were extracted with EA/TFA (99/1). Products were detected using selected positive ion monitoring of 307 for 2, 293 for 12, and 302 for 12 synthesized from [2-¹³C]-malonate. Traces i and ii show that both LovC and MlcG can work with LovB to produce 2 in the presence of 2 mM SAM; Trace iii: in the absence of SAM, LovC cannot reduce the tetraketide intermediate and shunt products 5-8 were synthesized (not shown). Trace iv: in the absence of SAM, MlcG can perform the enoylreduction and lead to the synthesis of 12; Trace v: when [2-¹³C]-malonate was used, the expected +9 mass increase was observed in 12 using LovB and MlcG. (C) The difference in substrate specificity between LovC and MlcG towards methylated and unmethylated tetraketide intermediate.