Polyketide natural products produced by bacteria and fungi are often characterized by the presence of multiple aromatic rings that are responsible for the activity of polyketides as both beneficial antibiotic and anticancer agents and as dangerous toxic compounds, such as the highly carcinogenic aflatoxins that are produced by fungal species from the Aspergillus family of molds. Polyketide ring formation by fungal enzymes called polyketide synthases (PKSs) is mediated by the enzyme's product template (PT) domain. However, the mechanism for aromatic ring formation from a linear intermediate with high fidelity has remained unclear. To reveal the cyclization mechanism, researchers at the University of California, Irvine, and The Johns Hopkins University solved the structure of the isolated PT domain of the PKS involved in aflatoxin production (PksA) to 1.8 Å using data from ALS Beamline 8.2.2 and SSRL Beamline 9-1. The crystal structure, along with biochemical studies, provides a paradigm for polyketide cyclization by fungal PKSs, an event that is necessary for imparting biological activity to this large class of clinically relevant natural products.

A megasynthase containing six enzyme domains covalently linked together, PksA catalyzes iterative reactions that produce a 20-carbon linear intermediate. At this point, if left unprotected, the highly reactive polyketide chain would undergo random cyclization to produce dead-end products. Instead, the linear intermediate enters PT, where it is stabilized and transformed into a specific bicyclic intermediate with a highly specific cyclization pattern exclusive to fungi, an amazing feat of origami. In contrast, bacteria fold the linear chain into a completely different pattern, as promoted by an enzyme called aromatase/cyclase (ARO/CYC) that shares no amino acid sequence similarity (homology) with PT. Therefore, although starting with the same linear chain, fungi and bacteria would fold the chain differently and end up producing completely different natural products.

The fungal polyketide synthase, PksA, is a single polypeptide that consists of six enzyme domains covalently linked together. It produces norsolorinic acid, the first isolatable intermediate in aflatoxin biosynthesis. The first three domains, SAT, KS, and MAT, along with ACP, function iteratively to synthesize a 20-carbon linear intermediate. Once the full-length, linear intermediate is formed, PT promotes an amazing feat of origami by folding the linear chain and cyclizing the chain between C4-C9 and C2-C11 carbons. The bicyclic intermediate is then passed on to TE where the third ring forms and the product is released.

The PT dimer crystal structure. One monomer is shown in surface representation (left) and the other in cartoon format (right). The surface representation highlights the single opening to the PT active site on each face of the dimer. In the cartoon monomer, the N-terminus is blue and the C-terminus is red. The long hydrophobic pocket is colored black.

The crystal structure of the isolated PT domain solved by the UC Irvine/Johns Hopkins team contains a dimer of double hot-dog folds reminiscent of ARO/CYC. Each monomer has an interior pocket, and the finite PT pocket length limits the possible substrate chain length while also orienting the linear polyketide, such that PT can selectively catalyze the cyclization of the linear chain. Two cocrystal structures were solved. In the first structure, a palmitic acid (a common fatty acid) molecule (fortuitously bound during E. coli purification) established the long, hydrophobic pocket and suggested that the 20-carbon intermediate enters the narrow PT pocket in a linear conformation anchored at the end of the pocket. The second structure, soaked with an analog of the bicyclic intermediate, showed that the chamber can accommodate two aromatic rings in a region in the middle of the chamber near the catalytic diad. Together with biochemical data, these structures confirmed the ability of PT to catalyze first- and second-ring cyclization/aromatization and established a general mechanism for fungal polyketide cyclization that has important implications for inhibitor design and controlled engineering of novel polyketides with altered cyclization patterns and possibly new bioactivities.

A close-up view of the PT active site in the cocrystal with the bicyclic substrate analog (HC8) bound. The hydrophobic binding region is filled with six waters (W1-W8). HC8 binds just below the catalytic His–Asp diad in a region called the cyclization chamber that is big enough to accommodate a bicyclic intermediate.

More generally, the fungal PT crystal structure, together with the ARO/CYC structure published by a UC Irivine group in 2008, has renewed the way we visualize polyketide cyclization. Although researchers had known for half a century that polyketides adopt special folds, the "streptomyces fold" for bacterial-generated polyketides and or "fungal fold" for fungal polyketides, no information has been available about how Nature folds and cyclizes a linear polyketide chain in such a highly specific manner. The ARO/CYC and PT structures revealed that regardless of bacterial or fungal PKS and despite low sequence homology, both ARO/CYC and PT have similar topology, and both have an interior pocket, whose size and shape directs the distinct ring patterns observed in bacterial and fungi.

A comparison of pocket shapes between the bacterial ARO/CYC and the fungal PT. Top: In bacteria, the tetracenomycin ARO/CYC folds a 20-carbon (C₂₀) polyketide to form the C9-C14 first ring. The substrate pocket (green mesh) is shallower, causing the polyketide to fold back on itself. Bottom: In fungi, the PksA PT folds a C₂₀ polyketide to form the C4-C9 first ring. The shape of the PT pocket (green mesh) is more extended, with the binding region at the end. The unique PT pocket shape keeps the bound substrate extended, while the C4-C9 first ring cyclization is mediated by hydrogen bonding between the substrate and the catalytic diad, resulting in a "kink" of the substrate between C4-C9 and their cyclization.

 

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Research conducted by J.M. Crawford, J.W. Labonte. A.L. Vagstad, E.A. Hill, and C.A. Townsend (Johns Hopkins University); and T.P. Korman, O. Kamari-Bidkorpeh, and S.-C. Tsai (University of California, Irvine).

Research funding: U.S. National Institutes of Health and the Damon Runyon Cancer Research Foundation. Operation of the ALS and SSRL are supported by the U.S. Department of Energy, Office of Basic Energy Sciences.

Publication about this research: J.M. Crawford, T.P. Korman, J.W. Labonte, A.L. Vagstad, E.A. Hill, O. Kamari-Bidkorpeh, S.-C. Tsai, C.A. Townsend, "Structural basis for biosynthetic programming of fungal aromatic polyketide cyclization," Nature 461, 1139 (2009).

ALSNews Vol. 307