Interrogation of Global Active Site Occupancy of a Fungal Iterative Polyketide Synthase Reveals Strategies for Maintaining Biosynthetic Fidelity

Abstract

Nonreducing iterative polyketide synthases (NR-PKSs) are responsible for assembling the core of fungal aromatic natural products with diverse biological properties. Despite recent advances in the field, many mechanistic details of polyketide assembly by these megasynthases remain unknown. To expand our understanding of substrate loading, polyketide elongation, cyclization, and product release, active site occupancy and product output were explored by Fourier transform mass spectrometry using the norsolorinic acid anthrone-producing polyketide synthase, PksA, from the aflatoxin biosynthetic pathway in Aspergillus parasiticus. Here we report the simultaneous observation of covalent intermediates from all catalytic domains of PksA from in vitro reconstitution reactions. The data provide snapshots of iterative catalysis and reveal an underappreciated editing function for the C-terminal thioesterase domain beyond its recently established synthetic role in Claisen/Dieckmann cyclization and product release. The specificity of thioesterase catalyzed hydrolysis was explored using biosynthetically relevant protein-bound and small molecule acyl substrates, and demonstrated activity against hexanoyl and acetyl, but not malonyl. Processivity of polyketide extension was supported by the inability of a nonhydrolyzable malonyl analog to trap products of intermediate chain lengths and by the detection of only fully extended species observed covalently bound to, and as the predominant products released by, PksA. High occupancy of the malonyl transacylase domain and fast relative rate of malonyl transfer compared to starter unit transfer indicate that rapid loading of extension units onto the carrier domain facilitates efficient chain extension in a manner kinetically favorable to ultimate product formation.

Rational reprogramming of fungal polyketide first-ring cyclization

“…This change would require radical rerouting of the polyketide substrate chain to expose a carbon to the catalytic base that is distal (S type) or proximal (F type) to the phosphopantetheine thioester. As shown above and observed previously by others (1,29,36), in trans reconstitution of iPKSs from dissected domains incurs a penalty for catalysis in terms of product yield and/or fidelity. Thus, we elected to conduct these experiments with nrPKSs where the heterologous PT domains replace their native equivalents in cis (29).…”

Modular Polyketide Synthases (PKSs): A New Model Fits All?

“…[21,22] Skiniotis and co-workers have presented a new model of PKSmediated natural compound synthesis based on moderate-resolution PikAIII data and biochemical studies. The new insight will change our view of PKSs.…”

The International Conference of Natural Product Biosynthesis (ICNPB, 8th US–Japan seminar on the Biosynthesis of Natural Products)

“…The conformation formed by SAT-KS-MAT and ACP domains was important for priming of hexanoyl groups and the number of condensation reactions of malonyl-CoAs to yield hexanoyl-primed octaketides. 39 In addition, TE/CLC was shown to have an editing role to cleave the thioesters of acetyl-ACP and hexanoyl-ACP, and decrease the amounts of derailment products derived from triketide intermediate. 39 Professor Hiroyuki Osada (RIKEN) presented the biosynthesis of reveromycin A (RM-A) and genetic engineering to produce various RM-A derivatives.…”

“…39 In addition, TE/CLC was shown to have an editing role to cleave the thioesters of acetyl-ACP and hexanoyl-ACP, and decrease the amounts of derailment products derived from triketide intermediate. 39 Professor Hiroyuki Osada (RIKEN) presented the biosynthesis of reveromycin A (RM-A) and genetic engineering to produce various RM-A derivatives. In vitro experiments showed that the post-PKS biosynthetic intermediates RM-A1a was converted to a dihydroketone intermediate by RevG, and the stereochemistry of the intermediate was controlled by RevJ, resulting in RM-A3a, which has a spiroacetal ring and shows 15S stereochemistry.…”