Molecular Characterization of the Cercosporin Biosynthetic Pathway in the Fungal Plant Pathogen Cercospora nicotianae.

Perylenequinones are a class of photoactivated polyketide mycotoxins produced by fungal plant pathogens that notably produce reactive oxygen species with visible light. The best-studied perylenequinone is cercosporin-a product of the Cercospora species. While the cercosporin biosynthetic gene cluster has been described in the tobacco pathogen Cercospora nicotianae, little is known of the metabolite's biosynthesis. Furthermore, in vitro investigations of the polyketide synthase central to cercosporin biosynthesis identified the naphthopyrone nor-toralactone as its direct product-an observation in conflict with published biosynthetic proposals. Here, we present an alternative biosynthetic pathway to cercosporin based on metabolites characterized from a series of biosynthetic gene knockouts. We show that nor-toralactone is the key polyketide intermediate and the substrate for the unusual didomain protein CTB3. We demonstrate the unique oxidative cleavage activity of the CTB3 monooxygenase domain in vitro. These data advance our understanding of perylenequinone biosynthesis and expand the biochemical repertoire of flavin-dependent monooxygenases.

Structural and Biochemical Analysis of Protein-Protein Interactions Between the Acyl-Carrier Protein and Product Template Domain.

In fungal non-reducing polyketide synthases (NR-PKS) the acyl-carrier protein (ACP) carries the growing polyketide intermediate through iterative rounds of elongation, cyclization and product release. This process occurs through a controlled, yet enigmatic coordination of the ACP with its partner enzymes. The transient nature of ACP interactions with these catalytic domains imposes a major obstacle for investigation of the influence of protein-protein interactions on polyketide product outcome. To further our understanding about how the ACP interacts with the product template (PT) domain that catalyzes polyketide cyclization, we developed the first mechanism-based crosslinkers for NR-PKSs. Through in vitro assays, in silico docking and bioinformatics, ACP residues involved in ACP-PT recognition were identified. We used this information to improve ACP compatibility with non-cognate PT domains, which resulted in the first gain-of-function ACP with improved interactions with its partner enzymes. This advance will aid in future combinatorial biosynthesis of new polyketides.

Systematic domain swaps of iterative, nonreducing polyketide synthases provide a mechanistic understanding and rationale for catalytic reprogramming.

Iterative, nonreducing polyketide synthases (NR-PKSs) are multidomain enzymes responsible for the construction of the core architecture of aromatic polyketide natural products in fungi. Engineering these enzymes for the production of non-native metabolites has been a long-standing goal. We conducted a systematic survey of in vitro "domain swapped" NR-PKSs using an enzyme deconstruction approach. The NR-PKSs were dissected into mono- to multidomain fragments and recombined as noncognate pairs in vitro, reconstituting enzymatic activity. The enzymes used in this study produce aromatic polyketides that are representative of the four main chemical features set by the individual NR-PKS: starter unit selection, chain-length control, cyclization register control, and product release mechanism. We found that boundary conditions limit successful chemistry, which are dependent on a set of underlying enzymatic mechanisms. Crucial for successful redirection of catalysis, the rate of productive chemistry must outpace the rate of spontaneous derailment and thioesterase-mediated editing. Additionally, all of the domains in a noncognate system must interact efficiently if chemical redirection is to proceed. These observations refine and further substantiate current understanding of the mechanisms governing NR-PKS catalysis.

Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis.

Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C-C ring closure reactions as opposed to thioester hydrolysis or O-C/N-C macrocyclization observed in previously reported TE structures. Catalysis of C-C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B(1), a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 A crystal structure displays an alpha/beta-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C-C cyclization. The biochemical studies, the 1.7 A crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C-C bond-forming class of TEs.

Quantitative PCR analysis of diepoxybutane and epihalohydrin damage to nuclear versus mitochondrial DNA.

The bifunctional alkylating agents diepoxybutane (DEB) and epichlorohydrin (ECH) are linked to the elevated incidence of certain cancers among workers in the synthetic polymer industry. Both compounds form interstrand cross-links within duplex DNA, an activity suggested to contribute to their cytotoxicity. To assess the DNA targeting of these compounds in vivo, we assayed for damage within chicken erythro-progenitor cells at three different sites: one within mitochondrial DNA, one within expressed nuclear DNA, and one within unexpressed nuclear DNA. We determined the degree of damage at each site via a quantitative polymerase chain reaction, which compares amplification of control, untreated DNA to that from cells exposed to the agent in question. We found that ECH and the related compound epibromohydrin preferentially target nuclear DNA relative to mitochondrial DNA, whereas DEB reacts similarly with the two genomes. Decreased reactivity of the mitochondrial genome could contribute to the reduced apoptotic potential of ECH relative to DEB. Additionally, formation of lesions by all agents occurred at comparable levels for unexpressed and expressed nuclear loci, suggesting that alkylation is unaffected by the degree of chromatin condensation.

Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function.

The polyketide synthase CTB1 is demonstrated to catalyze pyrone formation thereby expanding the known biosynthetic repertoire of thioesterase domains in iterative, non-reducing polyketide synthases.

DNA interstrand cross-linking by epichlorohydrin.

Epichlorohydrin (ECH), an important industrial chemical, is a bifunctional alkylating agent with the potential to form DNA cross-links. Occupational exposure to this suspect carcinogen leads to chromosomal aberrations, and ECH has been shown previously to undergo reaction with DNA in vivo and in vitro. We used denaturing polyacrylamide gel electrophoresis to monitor the possible formation of interstrand cross-links within DNA oligomers by ECH and the related compound, epibromohydrin (EBH). Although both compounds did indeed form cross-links between deoxyguanosine residues, EBH was a more efficient cross-linker than ECH. The optimal pH for cross-linking also varied, with ECH more efficient at pH 5.0 and EBH more efficient at pH 7.0. Both agents were relatively flexible in the sequences targeted, with comparable efficiencies for 5'-GGC and 5'GC sites. Furthermore, interstrand cross-linking by the two optical isomers of ECH correlated with their relative cytotoxicities, with R-ECH about twice as potent as S-ECH.