Deconstruction of iterative multidomain polyketide synthase function

Abstract

PksA, which initiates biosynthesis of the environmental carcinogen aflatoxin B1, is one of the multidomain iterative polyketide synthases (IPKSs), a large, poorly understood family of biosynthetic enzymes. We found that dissection of PksA and its reconstitution from selected sets of domains allows the accumulation and characterization of advanced octaketide intermediates bound to the enzyme, permitting the reactions controlled by individual catalytic domains to be identified. A product template (PT) domain unites with the ketosynthase and thioesterase in this IPKS system to assemble precisely seven malonyl-derived building blocks to a hexanoyl starter unit and mediate a specific cyclization cascade. Because the PT domain is common among nonreducing IPKSs, these mechanistic features should prove to be general for IPKS-catalyzed production of aromatic polyketides.

Fig. 1

Aflatoxin B₁ biosynthesis. (A) Biosynthetic pathway. PksA accepts a C₆ fatty acid from the associated fatty acid synthase subunits (HexA/HexB) and homologates acetyl units while attached to either the acyl carrier protein (ACP) or ketosynthase (KS) to a fixed chain length (octaketide), as shown in the hypothetical intermediate 1 (shown as the keto form), R = ACP or KS. PksA controls specific cyclization chemistry to release the norsolorinic acid anthrone (2), which is auto-oxidized in vitro to norsolorinic acid (3). 3 is processed through a series of oxidative rearrangements to aflatoxin B₁ (4). (B) Fungal nonreducing PKS domain architecture (PksA). Domains include SAT, KS, malonyl-CoA:ACP transacylase (MAT), PT, ACP, and thioesterase/Claisen cyclase (TE/CLC). Many known and apparently homologous enzymes that share similar domain organization synthesize an assortment of products with alternative chain lengths and cyclization scaffolds (5).

Fig. 2

Metabolite production from two-, three-, and four-part multi-domain combinations. (A) HPLC analysis. The enzymatic products were extracted into ethyl acetate, dried, and redissolved in dimethyl sulfoxide. The solution was diluted with mobile phase (80/20 acetoni-trile, 0.1% aqueous trifluoroacetic acid), and products were separated over a Prodigy octadecyl sulfate 3 column (100 Å, 5 μm, 4.6 × 250 mm; Phenomenex, Torrance, CA). The naphthopyrone 5 and norsolorinic acid anthrone (2) were observed at 280 nm, and norsolorinic acid (3) was observed at 310 nm. The boxed area at lower right shows the absorption at 280 nm of anthrone 2 amplified (50×). (B) Spontaneous versus enzymatic formation of ring C. The putative enzyme-bound intermediate 6 undergoes spontaneous chemistry in the absence of the TE/CLC domain to form the O-C cyclization product 5 or the C-C cyclization products 2 and 3. Addition of the TE/CLC, however, leads to the correct C-C cyclization products, confirming its role in catalyzing final cyclization and product release. The enzymatic anthrone product 2 auto-oxidizes to the anthraquinone 3.

Fig. 3

Chromophore formation. Enzymatic reactions were monitored over 15-min intervals for 240 min by UV-visible spectrophotometry. (A) SAT-KS-MAT control. All constructs by themselves similarly showed no change in absorbance. (B) Reconstitution of PksA. A 330-nm shoulder appears before reaching steady state in the presence of both tailoring domains (PT and TE/CLC). (C) PksA lacking the tailoring domains. Buildup of less highly conjugated chromophore(s) (~330 nm shoulder) occurs. (D) Chromophore formation in the presence of the PT domain. A 388-nm long-wavelength maximum from the formation of more highly conjugated chromophore(s) appears. Figure S3 shows spectra for all of the domain combinations, and the chromophore formation is highly similar for the same domain combinations whether they are tethered or not.

Fig. 4

Iterative intermediates. Relative intensities of acylated peptides from the ACP domain for reactions with the SAT-KS-MAT and either the ACP (A) or the PT-ACP (B). Mass spectrometric confirmation for the structure of each species is provided in figs. S5 to S9. Note that these 4-kD peptides bear different acyl intermediates, creating possible intensity biases stemming from differential ionization efficiencies during electrospray.