Animal FAS-like polyketide synthases produce diverse polypropionates

Abstract

Animal cytoplasmic fatty acid synthase (FAS) represents a unique family of enzymes that are classically thought to be most closely related to fungal polyketide synthase (PKS). Recently, a widespread family of animal lipid metabolic enzymes has been described that bridges the gap between these two ubiquitous and important enzyme classes: the animal FAS-like PKSs (AFPKs). Although very similar in sequence to FAS enzymes that produce saturated lipids widely found in animals, AFPKs instead produce structurally diverse compounds that resemble bioactive polyketides. Little is known about the factors that bridge lipid and polyketide synthesis in the animals. Here, we describe the function of EcPKS2 from Elysia chlorotica, which synthesizes a complex polypropionate natural product found in this mollusc. EcPKS2 starter unit promiscuity potentially explains the high diversity of polyketides found in and among molluscan species. Biochemical comparison of EcPKS2 with the previously described EcPKS1 reveals molecular principles governing substrate selectivity that should apply to related enzymes encoded within the genomes of photosynthetic gastropods. Hybridization experiments combining EcPKS1 and EcPKS2 demonstrate the interactions between the ketoreductase and ketosynthase domains in governing the product outcomes. Overall, these findings enable an understanding of the molecular principles of structural diversity underlying the many molluscan polyketides likely produced by the diverse AFPK enzyme family.

Keywords: metazoan biosynthesis; polyketide synthase; sacoglossan polypropionate.

Fig. 1.

AFPKs and their products. (A) The domain architecture of EcPKS1 and EcPKS2, products of EcPKS1 (1 and 2), and representative polyketides isolated from mollusks (3–8). (B) Phylogenetic analysis reveals that AFPKs are very similar to aFAS and only distantly related to PKSs. ⁰ indicates an inactive domain.

Fig. 2.

AFPK distribution in sacoglossan molluscs. (A) Phylogenetic tree of sacoglossan AFPKs, demonstrating that they form nine separate subclades (see SI Appendix, Fig. S4D for a larger version of this tree). (B) Comparison of AFPK KSs with polyketide chain length in the sacoglossans. Molluscs at right often contain longer chain products, reflected in the AFPK phylogeny, while those at left often contain shorter-chain products.

Fig. 3.

EcPKS2 synthesizes polypropionate precursors. MS chromatograms are shown, with the X axis as time in min and the Y axis being ion counts, analyzed using method a (SI Appendix). (A) EcPKS2 synthesizes the elysione (5) precursor (9) in the presence of 2 mM methylmalonyl-CoA and 2 mM NADPH. The Inset shows the same result, but in the presence of additional propionyl-CoA (2 mM). (B) EcPKS2 synthesizes tridachiahydropyrone precursors in the presence of 2 mM isovaleryl-CoA, 2 mM methylmalonyl-CoA, and 2 mM NADPH. While 9 was fully characterized by NMR, the other compounds in this figure were present in low abundance or were less stable, and thus were assigned using isotope labeling experiments and an application of EAD MS, which provides a large number of fragment ions for statistical analysis (SI Appendix). The small numbers present on each compound indicate the chain length from the initiating carboxyl group.

Fig. 4.

EcPKS2 has promiscuous selectivity for the starter unit. EcPKS2 reacts with 2 mM NADPH, 2 mM methylmalonyl-CoA, and: (A) 2 mM ethylmalonyl-CoA, and the Inset shows the same result when butyryl-CoA^a (2 mM) replace ethylmalonyl-CoA; (B) 2 mM acetyl-CoA^a; (C) 2 mM isobutyryl-CoA^a; (D) 2 mM benzoyl-CoA^a (Inset shows peaks that are closely overlapping in the main figure); (E) 16 mM benzoyl SNAC^b (Inset shows peaks that are closely overlapping in the main figure); (F) 16 mM nicotinoyl SNAC^b. MS chromatograms are shown, with the X axis as time in min and the Y axis as ion counts. ^a and ^b indicate different chemical analysis methods described in SI Appendix. Small numbers on structures are the carbon position indicating chain length.

Fig. 5.

MS chromatograms showing the major products when EcPKSf2-1 and EcPKSf1-2 are treated with methylmalonyl-CoA and NADPH.

Fig. 6.

Representative products from enzyme assays.