Polyketide synthase chemistry does not direct biosynthetic divergence between 9- and 10-membered enediynes Geoff P. Horsmana,1, Yihua Chena,1, Jon S. Thorsona,b, and Ben Shena,b,c,2 aDivision of Pharmaceutical Sciences, School of Pharmacy, bNational Cooperative Drug Discovery Group, and cDepartment of Chemistry, University of Wisconsin, Madison, WI 53705 Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved May 17, 2010 (received for review March 16, 2010) Enediynes are potent antitumor antibiotics that are classified as been sequenced, as exemplified by C-1027 (9), NCS (10), and 9- or 10-membered according to the size of the enediyne core maduropeptin (MDP) (11) for the 9-membered enediynes and structure. However, almost nothing is known about enediyne core CAL (12) and dynemicin (DYN) (13) for the 10-membered biosynthesis, and the determinants of 9- versus 10-membered en- enediynes, respectively (Fig. 1A). Common to all clusters is a five ediyne core biosynthetic divergence remain elusive. Previous work gene cassette (Fig. 1B, black) that encodes a unique enediyne- identified enediyne-specific polyketide synthases (PKSEs) that can specific iterative type I polyketide synthase (PKSE), a thioester- be phylogenetically distinguished as being involved in 9- versus ase (TE), and three additional genes of unknown function. The 10-membered enediyne biosynthesis, suggesting that biosynthetic PKSE generates an enediyne core precursor from iterative divergence might originate from differing PKSE chemistries. Recent condensation of malonyl-CoA (9); the high sequence identities in vitro studies have identified several compounds produced by the (41–68%, Table S1) and identical domain organization among PKSE and associated thioesterase (TE), but condition-dependent PKSEs (Fig. S1) suggest a common catalytic function and pro- product profiles make it difficult to ascertain a true catalytic differ- duct. These data have inspired speculation that PKSEs from both ence between 9- and 10-membered PKSE-TE systems. Here we re- classes generate an early common intermediate, which is directed port that PKSE chemistry does not direct 9- versus 10-membered to divergent fates by the action of 9- or 10-membered-specific enediyne core biosynthetic divergence as revealed by comparing accessory enzymes (Fig. 2A) (14). This hypothesis is consistent the products from three 9-membered and two 10-membered with the identification of 6–7 additional genes adjacent to the PKSE-TE systems under identical conditions using robust in vivo as- core cassette that are common only among the 9-membered gene says. Three independent experiments support a common catalytic clusters (Fig. 1B, gray). However, phylogenetic analysis of PKSEs function for 9- and 10-membered PKSEs by the production of a reveals that they group into two clades according to enediyne core i heptaene metabolite from: ( ) all five cognate PKSE-TE pairs in ring size. This ability to distinguish 9- versus 10-membered PKSEs Escherichia coli ii ;() the C-1027 and calicheamicin cognate PKSE- led to a predictive familial classification model for new enediyne- Streptomyces lividans iii TEs in K4-114; and ( ) selected native pro- encoding genes (14) and suggests that a possible catalytic differ- ducers of both 9- and 10-membered enediynes. Furthermore, PKSEs ence between the two PKSE families may be responsible for and TEs from different 9- and 10-membered enediyne biosynthetic generating the respective core structures. machineries are freely interchangeable, revealing that 9- versus Indeed, recent evidence for a functional distinction between 10-membered enediyne core biosynthetic divergence occurs 9- and 10-membered PKSEs is consistent with PKSE-directed beyond the PKSE-TE level. These findings establish a starting point biosynthetic divergence of the two enediyne families (Fig. 2B). for determining the origins of this biosynthetic divergence. For instance, we previously reported that NcsE and SgcE—the PKSEs involved in the biosynthesis of the 9-membered enediynes biosynthesis ∣ C-1027 ∣ thioesterase NCS and C-1027, respectively—produced heptaene (1) when co- expressed with their associated TEs, NcsE10 and SgcE10 (15, 16). nediyne natural products are some of the most potent antitu- In contrast, Liang and coworkers observed methyl hexaenone (2) Emor antibiotics discovered (1), and neocarzinostatin (NCS) as the major product of the in vitro combination of CalE8 and and calicheamicin (CAL) enjoy clinical success in Japan (2) CalE7, the PKSE-TE pair from the 10-membered enediyne and the United States (3), respectively. While their unique CAL (17). This apparently inefficient keto-reduction, or “ketor- mechanism of action (4, 5) and extraordinary biological activity eductase (KR) skipping,” by CalE8 in the final round of iterative generate interest among clinicians, the unusual molecular archi- BIOCHEMISTRY chain extension was therefore proposed as the first point of tecture of the enediynes motivates elucidation of the biosynthetic biosynthetic divergence between 9- and 10-membered enediyne logic directing their assembly. Most structurally remarkable is cores (Fig. 2B). the enediyne core, which is composed of two acetylenic groups However, subsequent reports using in vitro methods to deci- conjugated by a double bond within either a 9- or 10-membered pher functional differences between 9- and 10-membered PKSEs ring (Fig. 1A). Enediyne biosynthesis has been studied most reveal more complex behavior. For example, Townsend and cow- extensively for the 9-membered enediyne C-1027, revealing a 1 2 convergent process in which several peripheral moieties are orkers detected , , and several truncated polyketides from in appended to the enediyne core (6). Indeed, recent elucidation of peripheral pathway biochemistry has enabled rational design Author contributions: G.P.H., Y.C., and B.S. designed research; G.P.H. and Y.C. performed of novel enediyne analogs possessing altered biological activities research; G.P.H., Y.C., and B.S. analyzed data; J.T. contributed new reagents/analytic tools; (7, 8). However, despite rapid progress toward understanding and G.P.H., Y.C., and B.S. wrote the paper. peripheral moiety construction, the biosynthesis of the enediyne The authors declare no conflict of interest. core structure remains a mystery. This article is a PNAS Direct Submission. The enediynes are classified according to the enediyne core 1G.P.H. and Y.C. contributed equally to this work. ring size as either 9- or 10-membered, and how these two classes 2To whom correspondence should be addressed. E-mail: [email protected]. biosynthetically diverge is an intriguing question. Several biosyn- This article contains supporting information online at www.pnas.org/lookup/suppl/ thetic gene clusters for both 9- and 10-membered enediynes have doi:10.1073/pnas.1003442107/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1003442107 PNAS ∣ June 22, 2010 ∣ vol. 107 ∣ no. 25 ∣ 11331–11335 Downloaded by guest on September 24, 2021 Fig. 1. Selected 9- and 10-membered enediynes whose biosynthetic gene clusters have been sequenced. (A) Chemical structures of the 9- (C-1027, NCS, and MDP) and 10-membered (CAL, DYN, and ESP) enediynes with the enediyne core structures shown in bold. (B) Regions of enediyne gene clusters encoding enediyne core-biosynthesis with genes common among both 9- and 10-membered enediynes shown in black and genes common only among the 9-membered enediynes shown in gray. The pksE genes for 9- and 10-membered systems are named E and E8, respectively, while TE genes are named E10 and E7, respectively. vitro analysis of CalE8 and CalE7, indicating partial KR function We herein report that PKSE chemistry does not direct 9- versus in the final round of chain extension by CalE8 (dashed line in 10-membered enediyne core biosynthetic divergence (Fig. 2A) Fig. 2B). The variety of products was interpreted to be a conse- by comparing the products from three 9- (C-1027, NCS, MDP) quence of PKSE catalysis occurring in the absence of required and two 10-membered (CAL, DYN) PKSE-TE systems under accessory proteins and suggested that the apparent catalytic dif- identical conditions using robust in vivo assays. Three indepen- ference between 9- and 10-membered PKSEs may be an artifact dent experiments support a common catalytic function for 9- of assay conditions (18). Indeed, by optimizing in vitro reaction and 10-membered PKSEs by the production of 1 from: (i) all five conditions, Liang and coworkers variably observed 1, 2, and an cognate PKSE-TE pairs in E. coli;(ii) the SgcE-SgcE10 and unknown compound as major products from 10-membered CalE8-CalE7 cognate pairs in Streptomyces lividans K4-114; (CAL and DYN) and 9-membered (C-1027) PKSE-TE systems and (iii) selected native producers of both 9- and 10-membered (19). Although the assay could be modified to yield 1 as the major enediynes. Finally, as a first step toward investigating the possible product from each system, the condition-dependent product pro- role of protein–protein interactions in directing biosynthetic di- files of in vitro PKSE-TE systems caution against interpreting a vergence between 9- and 10-membered enediynes, we demon- common catalytic function for all PKSEs and motivate further strated no pathway-specific fidelity of the PKSE-TE interaction. study using more reproducible methods. These findings set the stage to further search for the origins of The difficulties with in vitro experimental systems for PKSE biosynthetic divergence