Identification of a starter unit acyl-carrier protein transacylase domain in an iterative type I polyketide synthase

Abstract

Polyketides are a class of natural products that exhibit a wide range of functional and structural diversity. They include antibiotics, immunosuppressants, antifungals, antihypercholesterolemics, and cytotoxins. Polyketide synthases (PKSs) use chemistry similar to fatty acid synthases (FASs), although building block variation and differing extents of reduction of the growing polyketide chain underlie their biosynthetic versatility. In contrast to the well studied sequential modular type I PKSs, less is known about how the iterative type I PKSs carry out and control chain initiation, elongation, folding, and cyclization during polyketide processing. Domain structure analysis of a group of related fungal, nonreducing PKSs has revealed well defined N-terminal domains longer than commonly seen for FASs and modular PKSs. Predicted structure of this domain disclosed a region similar to malonyl-CoA:acyl-carrier protein (ACP) transacylases (MATs). MATs play a key role transferring precursor CoA thioesters from solution onto FASs and PKSs for chain elongation. On the basis of site-directed mutagenesis, radiolabeling, and kinetics experiments carried out with individual domains of the norsolorinic acid PKS, we propose that the N-terminal domain is a starter unit:ACP transacylase (SAT domain) that selects a C(6) fatty acid from a dedicated yeast-like FAS and transfers this unit onto the PKS ACP, leading to the production of the aflatoxin precursor, norsolorinic acid. These findings could indicate a much broader role for SAT domains in starter unit selection among nonreducing iterative, fungal PKSs, and they provide a biochemical rationale for the classical acetyl "starter unit effect."

Fig. 1.

Related aromatic fungal metabolites. (A) Key intermediates in the aflatoxin B₁ biosynthetic pathway. The polyketide backbone 3 is cyclized by PksA to form the anthrone 4, and it is oxidized to the anthraquinone 2. Averufin (7) and sterigmatocystin (8) are isolable intermediates to aflatoxin B₁ (1). (B) Related pathways. Structures include emodinanthrone (5), emodin (6), dothistromin (9), YWA1 naphthopyrone (11), 1,3,6,8-tetrahydroxynaphthalene (10), aurofusarin (13), cercosporin (12), and bikaverin (14).

Fig. 2.

SAT alignment. (Upper) The SAT domain of PksA was aligned (ClustalX; ref. 51) with MATs of known structure from S. coelicolor (MAT-1, GenBank accession no. CAA60199) and E. coli (MAT-2, CAA77658) showing minimal local sequence similarity (22/34/23, 24/35/24, id/sim/gap, respectively). The secondary structure information and numbering are displayed for the S. coelicolor MAT. The serine and histidine comprising the catalytic dyad, as well as the glutamine in the oxyanion hole are indicated with asterisks. (Lower) SAT domains from several homologous fungal PKS enzymes that produce known natural products. Numbering is displayed for the SAT domain of PksA. Homologs include A. nidulans StcA (EAA61613) involved in the production of sterigmatocystin (8); Dothistroma septosporum PksA (PksA-2, AAZ95017), dothistromin (9); A. nidulans WA (Q03149), YWA1 naphthopyrone (11); Colletotrichum lagenarium PKS1 (BAA18956), 1,3,6,8-tetrahydroxynaphthalene (10); Gibberella zeae PKS12 (AAU10633), aurofusarin (13); and Cercospora nicotianae CTB1 (AAT69682), cercosporin (12). The conserved cysteine involved in transthioesterification is indicated with a diamond.

Fig. 3.

Starter unit transfer and domain organization. (A) Autoradiogram after SDS/PAGE to separate the ¹⁴C-transfer reactions. SAT (41.9 kDa), holo-ACP (20.8 kDa), SAT-C117A, and ACP-S1746A in different combinations were monitored for their ability to accept covalently the labeled hexanoyl starter unit. Lane 1, SAT; lane 2, holo-ACP; lane 3, SAT-C117A; lane 4, ACP-S1746A; lane 5, SAT/holo-ACP; lane 6, SAT/ACP-S1746A; lane 7, SAT-C117A/holo-ACP; and lane 8, SAT-C117A/ACP-S1746A. (B) Domain organization based on sequence similarity for HexA, HexB, and PksA. AT, acetyltransacylase; ER, enoyl reductase; DH, dehydrase; KR, ketoreductase; PT, product template; TE/CLC, thioesterase/Claisen cyclase. The proposed mechanism illustrating the selection and covalent attachment of the hexanoyl chain onto the SAT domain preparing the enzyme to load the PksA ACP is shown.

Fig. 4.

SAT transacylase rates (μmol of transfer per min/μmol of SAT) for malonyl-CoA, 0.6 ± 0.5; acetyl-CoA, 3.4 ± 0.7; butanoyl-CoA, 5.0 ± 0.3; hexanoyl-CoA, 30.4 ± 0.6; octanoyl-CoA, 14.2 ± 0.4; decanoyl-CoA, 0.2 ± 0.3; palmitoyl-CoA, 0.6 ± 0.5; and SAT-C117A hexanoyl-CoA, 1.2 ± 0.4.