Polyketide natural products constitute a broad class of compounds with diverse structural features and biological activities. Their biosynthetic machinery, represented by type I polyketide synthases, has an architecture in which successive modules catalyze two-carbon linear extensions and keto group processing reactions on intermediates covalently tethered to carrier domains. We employed electron cryo-microscopy to visualize a full-length module and determine sub-nanometer resolution 3D reconstructions that revealed an unexpectedly different architecture compared to the homologous dimeric mammalian fatty acid synthase. A single reaction chamber provides access to all catalytic sites for the intra-module carrier domain. In contrast, the carrier from the preceding module uses a separate entrance outside the reaction chamber to deliver the upstream polyketide intermediate for subsequent extension and modification. This study reveals for the first time the structural basis for both intra-module and inter-module substrate transfer in polyketide synthases, and establishes a new model for molecular dissection of these multifunctional enzyme systems.

The polyketide synthase (PKS) mega-enzyme assembly line uses a modular architecture to synthesize diverse and bioactive natural products that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents1. The architecture of a full-length PKS module from the pikromycin pathway creates a reaction chamber for the intra-module acyl carrier protein (ACP) domain that carries building blocks and intermediates between acyltransferase (AT), ketosynthase (KS), and ketoreductase (KR) active sites (see accompanying paper by Dutta et al.). Here we determined electron cryo-microscopy (cryo-EM) structures of a full-length PKS module in three key biochemical states of its catalytic cycle. Each biochemical state was confirmed by bottom-up liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC/FT-ICR MS). The ACP domain is differentially and precisely positioned after polyketide chain substrate loading on the active site of KS, after extension to the β-keto-intermediate, and after β-hydroxy product generation. The structures reveal the ACP dynamics for sequential binding to catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. During the enzymatic cycle the KR domain undergoes dramatic conformational rearrangements that enable optimal positioning for reductive processing of the ACP-bound polyketide chain elongation intermediate. These findings have crucial implications for the design of functional PKS modules, and for the engineering of pathways to generate pharmacologically relevant molecules.

The ICF syndrome (i̲mmunodeficiency, c̲entromeric region instability, f̲acial anomalies) is a unique DNA methylation deficiency disease diagnosed by an extraordinary collection of chromosomal anomalies specifically in the vicinity of the centromeres of chromosomes 1 and 16 (Chr1 and Chr16) in mitogen-stimulated lymphocytes. These aberrations include decondensation of centromere-adjacent (qh) heterochromatin, multiradial chromosomes with up to 12 arms, and whole-arm deletions. We demonstrate that lymphoblastoid cell lines from two ICF patients exhibit these Chr1 and Chr16 anomalies in 61% of the cells and continuously generate 1qh or 16qh breaks. No other consistent chromosomal abnormality was seen except for various telomeric associations, which had not been previously noted in ICF cells. Surprisingly, multiradials composed of arms of both Chr1 and Chr16 were favored over homologous associations and cells containing multiradials with 3 or >4 arms almost always displayed losses or gains of Chr1 or Chr16 arms from the metaphase. Our results suggest that decondensation of 1qh and 16qh often leads to unresolved Holliday junctions, chromosome breakage, arm missegregation, and the formation of multiradials that may yield more stable chromosomal abnormalities, such as translocations. These cell lines maintained the abnormal hypomethylation in 1qh and 16qh seen in ICF tissues. The ICF-specific hypomethylation occurs in only a small percentage of the genome, e.g., ICF brain DNA had 7% less 5-methylcytosine than normal brain DNA. The ICF lymphoblastoid cell lines, therefore, retain not only the ICF-specific pattern of chromosome rearrangements, but also of targeted DNA hypomethylation. This hypomethylation of heterochromatic DNA sequences is seen in many cancers and may predispose to chromosome rearrangements in cancer as well as in ICF.

The biocatalytic transformations used by chemists are often restricted to simple functional-group interconversions. In contrast, nature has developed complexity-generating biocatalytic reactions within natural product pathways. These sophisticated catalysts are rarely employed by chemists, because the substrate scope, selectivity and robustness of these catalysts are unknown. Our strategy to bridge the gap between the biosynthesis and synthetic chemistry communities leverages the diversity of catalysts available within natural product pathways. Here we show that, starting from a suite of biosynthetic enzymes, catalysts with complementary substrate scope as well as selectivity can be identified. This strategy has been applied to the oxidative dearomatization of phenols, a chemical transformation that rapidly builds molecular complexity from simple starting materials and cannot be accomplished with high selectivity using existing catalytic methods. Using enzymes from biosynthetic pathways, we have successfully developed a method to produce ortho-quinol products with controlled site- and stereoselectivity. Furthermore, we have capitalized on the scalability and robustness of this method in gram-scale reactions as well as multi-enzyme and chemoenzymatic cascades.

Drug‐induced DNA demethylation in normal human cells and inherited localized hypomethylation in mitogen‐stimulated lymphocytes from patients with a rare recessive disease (ICF: i―mmunodeficiency, c―entromeric region instability, f―acial anomalies) are associated with karyotypic instability. This chromosomal recombination is targeted to heterochromatin in the vicinity of the centromere (pericentromeric region) of human chromosome 1. Pericentromeric rearrangements in this chromosome as well as overall genomic hypomethylation are frequently observed in many kinds of cancer, including breast adenocarcinoma. We found that almost half of 25 examined breast adenocarcinomas exhibited hypomethylation in satellite 2 DNA, which is located in the long region of heterochromatin adjacent to the centromere of chromosome 1 and is normally highly methylated. One of the 19 examined non‐malignant breast tissues displaying fibrocystic changes was similarly hypomethylated in this satellite DNA. We also looked at an opposing type of methylation alteration in these cancers, namely, hypermethylation in a tumor‐suppressor gene region that is frequently hypermethylated in breast cancers. We found that increased methylation in the E‐cadherin promoter region and decreased methylation in satellite 2 DNA were often present in the same breast cancers. While hypermethylation in certain tumor‐suppressor gene regions may favor tumorigenesis by repressing transcription, demethylation of other DNA sequences may predispose to cancer‐promoting chromosomal re‐arrangements. Int. J. Cancer 77:833–838, 1998. © 1998 Wiley‐Liss, Inc.