Biochemical characterization of the minimal domains of an iterative eukaryotic polyketide synthase

Abstract

Iterative type I polyketide synthases (PKS) are megaenzymes essential to the biosynthesis of an enormously diverse array of bioactive natural products. Each PKS contains minimally three functional domains, β-ketosynthase (KS), acyltransferase (AT), and acyl carrier protein (ACP), and a subset of reducing domains such as ketoreductase (KR), dehydratase (DH), and enoylreductase (ER). The substrate selection, condensation reactions, and β-keto processing of the polyketide growing chain are highly controlled in a programmed manner. However, the structural features and mechanistic rules that orchestrate the iterative cycles, processing domains functionality, and chain termination in this kind of megaenzymes are often poorly understood. Here, we present a biochemical and functional characterization of the KS and the AT domains of a PKS from the mallard duck Anas platyrhynchos (ApPKS). ApPKS belongs to an animal PKS family phylogenetically more related to bacterial PKS than to metazoan fatty acid synthases. Through the dissection of the ApPKS enzyme into mono- to didomain fragments and its reconstitution in vitro, we determined its substrate specificity toward different starters and extender units. ApPKS AT domain can effectively transfer acetyl-CoA and malonyl-CoA to the ApPKS ACP stand-alone domain. Furthermore, the KS and KR domains, in the presence of Escherichia coli ACP, acetyl-CoA, and malonyl-CoA, showed the ability to catalyze the chain elongation and the β-keto reduction steps necessary to yield a 3-hydroxybutyryl-ACP derivate. These results provide new insights into the catalytic efficiency and specificity of this uncharacterized family of PKSs.

Keywords: PKS biochemistry; domain deconstruction; iterative PKS; substrate specificity.

Figure 1

Phylogeny of polyketide synthases. Maximum likelihood tree based on KS‐AT didomain from representative type I PKS families including iterative and modular bacterial PKSs, NR, and highly reducing fungal PKSs, metazoan PKSs (birds and reptiles), and fatty acids syntases (birds, reptiles and human). Bootstrap values (based on 1000 replicates) are indicated at the tree nodes. The scale bar below denotes substitutions per site.

Figure 2

ApPKS deconstruction. (A) Schematic representation of ApPKS domain organization and all the constructions used in this study. (B) Coomasie‐stained SDS/PAGE of purified constructs after Ni²⁺ affinity purification. Lane 1, KS (47.1 KDa), lane 2, KS‐AT (97.6 KDa), lane 3, KR (87.8 KDa), lane 4, ACP1 (7.8 KDa), lane 5, ACP2 (13.6 KDa), and lane 6, ACP3 (20.9 KDa). All constructs were expressed as N‐terminal 6xHis‐tag fusion proteins with the exception of KS, which was expressed as C‐terminal his‐tag fusion. The ACP constructs were fusions to 6xHis‐thiorredoxin (trx; Table S1). TEV represent the TEV protease cleavage site and PAT stands for post‐AT Linker. In lanes 1, 2, and 3, the protein at 60 KDa corresponds to the chaperon GroEL obtained during the protein purification as by‐product. (C) LC‐MS chromatogram of the peptides from Apo and Holo forms of ACP1 after digestion with Trypsin and GluC. (D) Mass spectra of the Apo and Holo forms of ACP1 peptides, the ions shown correspond to the most abundant ion formed, (M + 3H⁺)³⁺.

Figure 3

Acylation and transacylation of the KS‐AT didomain with different starter units. (A) Schematic representation of the transfer reaction of starter or extender units to ACP1 by KS‐AT. The starter units analyzed were acetyl‐CoA and propionyl‐CoA, and the extender units were malonyl‐CoA and methylmalonyl‐CoA. (B) SDS/PAGE autoradiography of purified KS‐AT and ACP1 coincubated with radiolabeled acetyl‐CoA or propionyl‐CoA. (C) Mass spectrometry analysis of the ACP formed in transacylations assay with acetyl‐CoA. Left panel, mass spectra of the ACP peptide bound to an acetyl group. The most abundant specie found was (M + 3H⁺)³⁺. Right panel, pantetheinyl ejection fragments observed during tandem mass spectrometry of the ion at m/z 969.14, the most abundant ion is at m/z 303.13, the pantetheinyl elimination of acetyl‐ACP. (D) Mass spectrometry analysis of the ACP formed in transacylations assay with propionyl‐CoA. Left panel, mass spectra of the ACP peptide bound to a propionyl group. The most abundant specie found was (M + 3H⁺)³⁺. Right panel, pantetheinyl ejection fragments observed during tandem mass spectrometry of the ion at m/z 973.80, the most abundant ion is at m/z 317.15, the pantetheinyl elimination of propionyl‐ACP.

Figure 4

Acylation and transacylation of KS‐AT didomain with different extender units. (A) SDS/PAGE autoradiography of KS‐AT and ACP1 coincubated with radiolabeled malonyl‐CoA and methylmalonyl‐CoA. (B) Mass spectrometry analysis of the ACP formed in transacylation assays with malonyl‐CoA. Left panel, mass spectra of the ACP peptide bound to an acetyl group. The most abundant species found was (M + 3H⁺)³⁺. Right panel, pantetheinyl ejection fragments observed during tandem mass spectrometry of the ion at m/z 983.79, the ion at m/z 347.14 belongs to the pantetheinyl elimination of malonyl‐ACP. The high signal for ion at m/z 303.14 belongs to pantetheinyl elimination of acetyl‐ACP formed by decarboxylation induced by the collisional energy applied for MS/MS. (C) Mass spectrometry analysis of the ACP formed in transacylations assay with methylmalonyl‐CoA. Left panel, mass spectra of the ACP peptide bound to an acetyl group. The most abundant species found was (M + 3H⁺)³⁺. Right panel, pantetheinyl ejection fragments observed during tandem mass spectrometry of the ion at m/z 988.48, the ion at m/z 361.13 is the pantetheinyl elimination of methylmalonyl‐ACP. The high signal for ion at m/z 317.15 belongs to pantetheinyl elimination of propionyl‐ACP formed by decarboxylation induced by the collisional energy applied for MS/MS. (D) Conformational sensitive gel electrophoresis of the transacylation assay performed with KS‐AT and KS‐AT₀. (*) lane 2: self‐malonylation of ACP1.

Figure 5

Kinetic studies of the AT‐catalyzed reactions. (A) Michaelis–Menten plots of the AT catalyzed reactions: hydrolysis (blue) and acyl transfer to ACP1 (red) of the indicated substrates. The experimental assays for hydrolytic reactions were performed under the same condition of transfer reactions but in the absence of ACP1 acceptor. Error bars reflect the standard deviation between three biological replicates. (B) The kinetic parameters listed in the table were obtained by varying the concentration of the acyl‐CoA used as substrate. All the kinetic constants listed in the table are apparent.

Figure 6

Condensation activity of KS domain. (A) Schematic representation of the condensation reaction where the acetyl group bound to the KS domain and the malonyl group bound to the ACP form the new carbon–carbon bond by Claisen condensation, given 3‐ketobutyryl‐ACP. The KR reduces this product to form 3‐hydroxybutyryl‐ACP. (B) SDS/PAGE autoradiograph of the ApPKS KS domain coincubated with radiolabeled acetyl‐CoA and malonyl‐CoA, respectively. KS can be acylated with acetyl‐CoA and to a lesser extent with malonyl‐CoA. Labeling of KS domain with malonyl‐CoA probably suggests that the KS domain may decarboxylate malonyl‐CoA or that a spountaneous decarboxylation occurs (remaining an acetyl group attached to the Cys of the active site). (C) Thin layer chromatography autoradiograph of the condensation products after alkaline hydrolysis. (D) GC‐MS analysis of the condensation products. After alkaline hydrolysis, the ACP released species which were silylated and separated by gas chromatography. Upper panel; chromatogram of a silylated 3‐hydroxybutyric acid standard. Middle panel; reaction without the KS, where no product is formed and only the substrate malonic acid is present. Lower panel; reaction where the condensation product is formed. The chemical structures of the substrate and product trimethylsilyl derivates are below the chromatographic peaks.

Figure 7

Analysis of the condensation reaction. (A) Thin layer chromatography autoradiograph after alkaline hydrolysis of reactions containing KS or KS‐AT as condensing domains. The expected product, 3‐hydroxy butyrate, and the side product, acetate, are indicated. (B) Thin layer chromatography autoradiograph after alkaline hydrolysis of reactions containing acetyl‐ and propionyl‐SNAC as starter units. The expected condensation products are 3‐hydroxy butyrate in lane 1 and 3‐hydroxy pentanoate in lane 3.