EncM, a versatile enterocin biosynthetic enzyme involved in Favorskii oxidative rearrangement, aldol condensation, and heterocycle-forming reactions Longkuan Xiang*, John A. Kalaitzis*, and Bradley S. Moore*†‡ *Department of Pharmacology and Toxicology, College of Pharmacy, and †Department of Chemistry, University of Arizona, Tucson, AZ 85721 Edited by Stephen J. Benkovic, Pennsylvania State University, University Park, PA, and approved September 27, 2004 (received for review July 29, 2004) The bacteriostatic natural product enterocin from the marine rangement prevents successive cyclizations by means of aldol microbe ‘‘Streptomyces maritimus’’ has an unprecedented carbon condensations to characteristic multiaromatic end products. The skeleton that is derived from an aromatic polyketide biosynthetic branched intermediate gives rise not only to the tricyclic caged pathway. Its caged tricyclic, nonaromatic core is derived from a core of enterocin, but also, after decarboxylation, to the struc- linear poly-␤-ketide precursor that formally undergoes a Favorskii- turally diverse wailupemycins A to C (2–4) (8). like oxidative rearrangement. In vivo characterization of the gene Biosynthetic carbon skeletal rearrangements involving oxida- encM through mutagenesis and heterologous biosynthesis dem- tive Favorskii-like chemistry have been proposed in only a few onstrated that its protein product not only is solely responsible for secondary metabolic pathways on the basis of feeding experi- the oxidative C—C rearrangement, but also facilitates two aldol ments with isotopically labeled biosynthetic intermediates. Ad- condensations plus two heterocycle forming reactions. In total, at ditional examples include the fungal polyketide aspyrone (9) and least five chiral centers and four rings are generated by this the dinoflagellate polyether okadaic acid (10). The recent clon- multifaceted flavoprotein. Heterologous expression of the ente- ing and sequencing of the enterocin biosynthetic gene cluster in rocin biosynthesis genes encABCDLMN in Streptomyces lividans the marine isolate ‘‘Streptomyces maritimus’’ has now allowed us resulted in the formation of the rearranged metabolite desmethyl- to examine this biosynthetic reaction in greater detail (5). 5-deoxyenterocin and the shunt products wailupemycins D–G. Mutational analysis revealed that the putative FAD-dependent Addition of the methyltransferase gene encK, which was previ- oxygenase EncM and the methyltransferase EncK jointly cata- ously proposed through mutagenesis to additionally assist EncM in lyze the rearrangement because inactivation of either encoding the Favorskii rearrangement, shifted the production to the O- gene terminated enterocin (1) production and caused the accu- methyl derivative 5-deoxyenterocin. The O-methyltransferase mulation of the nonrearranged and nonmethylated polyketides EncK seems to be specific for the pyrone ring of enterocin, because wailupemycins D to G (5–8) (Fig. 1) (7). This result suggested bicyclic polyketides bearing pyrone rings are not methylated in that the rearrangement could be radical in nature, with EncK vivo. Expression of encM with different combinations of homolo- providing two biosynthetic functions, first as a radical source and gous actinorhodin biosynthesis genes did not result in the produc- second as a source of the O-methyl group. In this study, we set tion of oxidatively rearranged enterocin–actinorhodin hybrid com- out to further examine this highly unusual biosynthetic rear- pounds as anticipated, suggesting that wild-type EncM may be rangement in a series of heterologous expression experiments. specific for its endogenous type II polyketide synthase or for benzoyl-primed polyketide precursors. Materials and Methods General Experimental Details. NMR spectra were recorded on ͉ ͉ ͉ flavoprotein methyltransferase polyketide synthase Streptomyces Bruker (Billerica, MA) DRX-300 and DRX-600 spectrometers. 1H and 13C chemical shifts were referenced to the solvent peak ulticyclic aromatic polyketides such as the clinically im- (DMSO-d6) ␦ 2.49 and 39.5 ppm, respectively. Standard param- Mportant tetracyclines, anthracyclines, and angucyclines are eters were used for 1D and 2D NMR spectra obtained, which biosynthesized in actinomycetes on heterodimeric type II included 1H, 13C, heteronuclear multiple quantum correlation polyketide synthases (PKSs) (1, 2). These dissociable complexes (HMQC), and heteronuclear multiple-bond correlation of monofunctional enzymes contain a ‘‘minimal’’ set of proteins (HMBC). Optical rotation was measured on a Jasco (Easton, [the two ketosynthase subunits KS␣ and KS␤ (also referred to as MD) P-1010 polarimeter. A Waters 600 pump equipped with a the chain length factor; ref. 3), acyl carrier protein (ACP), and Waters 2487 dual ␭ absorbance detector was used for semi- malonyl-CoA:ACP acyltransferase] that are required for the preparative HPLC separations. A Waters 996 photodiode array BIOCHEMISTRY biosynthesis of the polyketide chain. Additional PKS subunits, detector was used for analytical HPLC. High-resolution fast including ketoreductases (KR), cyclases, aromatases, oxygen- atom bombardment MS (HRFABMS) were recorded on a JEOL ases, etc., are responsible for modification of the nascent poly- HX110A high resolution mass spectrometer at the Mass Spec- ␤ -carbonyl intermediate to form the aromatic product. The trometry Facility, Department of Chemistry, University of context-dependent nature of the type II PKS-encoding gene set Arizona. thus dictates the metabolic outcome of the pathway, which is dominated by planar, polyaromatic natural products. Bacterial Strains and General Techniques for DNA Manipulation. The bacteriostatic agent enterocin (1), however, stands apart Streptomyces lividans K4-114 was used as a host for transforma- from all other type II PKS-derived polyketides (4). Enterocin tion of plasmids (11). Escherichia coli XL1-Blue was used for the does not contain a planar polyaromatic core structure, but rather uniquely has a nonaromatic caged core resulting from a branched intermediate. Although the early stages of enterocin This paper was submitted directly (Track II) to the PNAS office. biosynthesis have been shown to proceed by means of a typical Abbreviations: ACP, acyl carrier protein; KR, ketoreductase; PKS, polyketide synthase; aromatic polyketide pathway (5) (Fig. 1), labeling (6) and genetic HMBC, heteronuclear multiple-bond correlation; HRFABMS, high-resolution fast atom (7) experiments revealed that its biosynthesis involves an un- bombardment MS. precedented oxidative rearrangement of its nascent poly-␤- ‡To whom correspondence should be addressed. E-mail: [email protected]. carbonyl intermediate. This carbon skeletal Favorskii-like rear- © 2004 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0405508101 PNAS ͉ November 2, 2004 ͉ vol. 101 ͉ no. 44 ͉ 15609–15614 Downloaded by guest on September 24, 2021 Fig. 1. Proposed biosynthetic pathway to enc-based polyketides (1–10) and structures of analogous act-derived polyketides (11-13). Path A involves monooxidation of 14 to the trione intermediate 16, whereas path AЈ involves a dioxygenase role of EncM in which the C7–C12 olefin of the aldol product 15 is oxidatively cleaved to 16. The Favorskii-like rearrangement is speculated to occur on a putative ACP (EncC)-bound intermediate. The relative size of the reaction arrows reflects the flux in the pathway to the major product 1 as can be observed chromatographically (see Fig. 2). Dashed lines in structures 16 and 17 represent newly created bonds. In compounds 14–17, the carbon numbering is based on linear 14 in which priority is given to the carboxyl carbon; the numbering in compounds 1, 9, and 10 is in contrast based on that defined in ref. 18 for 9. manipulation of plasmid DNA. Streptomyces coelicolor A3(2) gent act promoters was cloned into analogous sites of pMP6 to was used to extract genomic DNA for cloning act genes, and the create pBM43. cosmid clone pJP15F11 (5), which harbors the entire enc bio- pBM47 is a pSEK4 derivative in which a 6.1-kb PacI͞EcoRI synthesis gene cluster, was used as the source of DNA for cloning fragment harboring actI-ORFsI,II,III and encMN was cloned into enc genes. The E. coli–Streptomyces shuttle pSEK4 and pRM5- the analogous sites. A 3.0-kb fragment containing actI- based vectors were used for all expression experiments in ORFsI,II,III was PCR amplified from S. coelicolor A3 (2) Streptomyces (3). Recombinant DNA procedures were per- genomic DNA by using primers 5Ј-GCATTAATTAAGCG- formed by standard techniques (12, 13). Restriction enzyme- GCGGTCGAAGGGAGATG-3Ј (forward, PacI underlined) digested DNA fragments were recovered from agarose gels with and 5Ј-GCCTCTAGACCACCCGGTGTTCTCCCGG-3Ј (re- the QIAquick DNA Purification Kit (Qiagen, Valencia, CA). verse, XbaI underlined) and cloned into pCR-Blunt to create Oligonucleotides were obtained from Sigma Genosys. PCR was pBM44. The 3.0-kb XbaI-HindIII fragment harboring actI- carried out on a PTC-2000 Peltier thermal cycler (MJ Research, ORFsI,II,III from pBM44 was ligated into the analogous sites of Cambridge, MA) with PfuTurbo (Stratagene) DNA polymerase. pBM45 to create pBM46. pBM45 is a pCR-Blunt-derived plas- DNA sequencing by BigDye terminator cycle sequencing reac- mid in which an XbaI͞NheI 3.1-kb fragment containing encMN tion using an ABI 377 sequencer (Applied Biosystems) was was cloned into the analogous sites. encMN was PCR amplified performed at the Laboratory of