Point of
VIEW
Clearing the Skies over Modular Polyketide Synthases David H. Sherman†,‡,§,* and Janet L. Smith¶,*
† Departments of Medicinal Chemistry, ‡Microbiology and Immunology, §Chemistry, and ¶Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109
M
acrolide antibiotics are among the most effective and successful natural product anti-infective agents and are featured in ongoing efforts toward the development of new therapeutics that target drug-resistant pathogens. Erythromycin was the first macrolide to be introduced into human use, and its structure continues to be a crucial template for production of semisynthetic antibiotics, particularly the new ketolide anti-infective agents introduced recently into the clinic (1). The erythromycin biosynthetic pathway (2, 3) has also been a key model system for understanding the intricate series of steps involved in assembly of the 14-membered macrolactone ring system and the glycoside appendages that together form the macrolide class of antibiotic agents. Assembly of the core macrolactone of erythromycin is prescribed by a series of multifunctional enzymes called modular polyketide synthases (PKSs), which have been studied genetically and biochemically over the past 15 yr. The frontier in new efforts to understand and engineer these fascinating megaenzymes requires detailed atomiclevel structures of the individual catalytic domains and domain partners that comprise the modular PKS system. Recently, Tang et al. (4) provided penetrating new insights into the workings of this biochemical assembly line in reporting the first crystal structure of a modular PKS ketoacyl synthase-AT di-domain. Together with a recent crystal structure of a modular PKS
www.acschemicalbiology.org
ketoreductase (KR) domain (5) and crystallographic analyses of mammalian and fungal fatty acid synthases (6, 7), a unified and unexpected picture is emerging that offers a refined understanding of the versatile capabilities of these extraordinary biological catalysts. Since the first studies of polyketide biosynthesis a century ago (8), chemists have recognized the striking parallels between mechanisms involving assembly of this hugely diverse family of bioactive natural products and their cousins, the more chemically modest, but essential, fatty acids (9). Both are constructed by linking a shortchain acyl-coenzyme A (CoA) starter unit with several additional short-chain acyl-CoA extender units to form a more complex product. For both polyketides and fatty acids, the center of this process is the set of carbon–carbon bond-forming reactions that link the acyl subunits, with both systems depending on an acyl carrier protein (ACP) to mediate the reaction sequences for chain elongation and subsequent keto group processing. In terms of structural outcome, the similarities between polyketide metabolites and fatty acids stop here. The products of the fatty acid biosynthetic machinery (fatty acid synthase or FAS) are limited with respect to chain length, involve a small number of biosynthetic subunits or appended functional groups, and usually result in a metabolic product devoid of stereochemistry. By contrast, enzymes involved in construction of complex polyketides (the
A B S T R A C T Modular polyketide synthases (PKSs) are large multifunctional proteins that synthesize complex polyketide metabolites in microbial cells. A series of recent studies confirm the close protein structural relationship between catalytic domains in the type I mammalian fatty acid synthase (FAS) and the basic synthase unit of the modular PKS. They also establish a remarkable similarity in the overall organization of the type I FAS and the PKS module. This information provides important new conclusions about catalytic domain architecture, function, and molecular recognition that are essential for future efforts to engineer useful polyketide metabolites with valuable biological activities.
*Corresponding authors,
[email protected],
[email protected].
Published online September 15, 2006 10.1021/cb600376r CCC: $33.50 © 2006 by American Chemical Society
VOL.1 NO.8 • ACS CHEMICAL BIOLOGY
505
Figure 1. Comparison of type I FAS and modular PKS. a) Mammalian FAS functions iteratively to generate a long-chain saturated fatty acid chain. b) Bacterial modular PKS, derived from primordial bacterial type I FAS (that in turn evolved into mammalian FAS) is composed of a series of modules bearing specific combinations of catalytic domains that operate sequentially. The metabolic outcome is a complex linear polyketide chain elongation intermediate that is finally cyclized by the terminal TE domain. c) Examples of polyketide chemical diversity, including (from left to right) epothilone, avermectin, rapamycin, and erythromycin aglycone.
modular PKSs) are renowned for their ability to create chemical diversity involving starter and extender units, regio- and stereochemistry, and chain length. An elaborate “programming” process that resides within the basic synthase unit, or PKS module, enables this diversity. A fundamental enigma exists when comparing the basic enzymatic subunit of a type I FAS with a fully equipped type I PKS module (Figure 1). Both are huge multifunctional proteins composed of an equal set of 506
VOL.1 NO.8 • 505–509 • 2006
catalytic domains, including a keto acyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoylreductase (ER), KR, ACP, and terminal thioesterase (TE). In the case of FAS, these catalytic domains exist on a single multifunctional enzyme that operates iteratively, where the growing fatty acid chain cycles through the prescribed set of catalytic reactions until the proper chain length is achieved and is finally off-loaded by the terminal TE. In the modular PKSs, this same series of seven catalytic domains (KS-AT-KRSHERMAN AND SMITH
DH-ER-ACP-(TE)) participates as part of a larger composite complex involving many modules that include sequential chain extensions and keto group processing reactions, where the growing acyl chain is passed from one module to another until it is off-loaded and (typically) cyclized as a macrolactone by the terminal TE (10). From this more complex arrangement, polyketide metabolites can display an almost limitless chemical diversity. The PKS module and the type I FAS have a clear evolutionary connecwww.acschemicalbiology.org
Point of
VIEW tion, apparent both in the similar sequences of their catalytic domains and in the order of these domains in the multifunctional proteins. However, the wide distribution of PKSs in the microbial world and the extreme chemical diversity of their products are the result of considerable divergence from their common FAS ancestor (Figure 1). Nature has removed catalytic domains from many modules, rendered domains inactive in others, rearranged domains in some, and occasionally imported non-FAS catalytic domains (e.g., the curacin (11) and jamaicamide (12) PKSs). The linking sequences in PKS modules and FAS appear dissimilar, in contrast to the sequences of the catalytic domains that they connect. In light of such divergence, what relationship, if any, remains in the overall structures of the PKS module and the FAS? Over the past few months, several crystal structures have been reported that focus on distinct aspects of these multifunctional proteins. Three seemingly disparate contributions (4–6) provide a new, unifying view of the FAS and PKS systems (Figure 2). In reality, the great divergence of PKS modules has clouded the fundamental similarity in overall architecture of the PKS module and its type I FAS ancestor. The nearly simultaneous publication of these three new crystal structures clears the clouds, sheds a brilliant light on their structure and mechanisms, provides directions for additional studies, and reminds us once again that protein structure is far more conserved than primary sequence. First to appear was the structure of the porcine FAS from Ban and coworkers (6). Crystallization of this 540-kDa dimeric megaenzyme was a major achievement. Although the resolution of the electron density map was too low (4.5 Å) for detailed fitting of the amino acid sequence, previously reported structures of five enzymes (KS, AT, DH, ER, KR) could be fit to the map with a high degree of confidence. The overall structure has upper and lower halves www.acschemicalbiology.org
connected by a narrow “waist” (Figure 2). The KS and AT domains reside in the lower half of the protein, and the DH, ER, and KR domains reside in the upper half. The fundamentally dimeric KS, DH, and ER domains reside along the dimer axis, and the monomeric AT and KR domains are at the periphery. Even after all the catalytic domains had been placed, some regions of electron density were unaccounted for. These were ascribed to long linking sequences (between KS and AT domains, between DH and ER domains) and to the terminal TE. One month later, Keatinge-Clay and Stroud (5) reported the structure of the KR domain from module 1 of the erythromycin PKS. Crystals were derived from a tryptic fragment that included a large linker region preceding the assigned KR domain, as well as nearly 100 residues following it. The structure contained a huge surprise; the entire region forms a single protein structure consisting of an amino-terminal “structural” domain intimately associated with the previously assigned catalytic domain at the carboxy terminus. It does not appear that the KR catalytic domain can exist in the absence of its structural domain partner. Keatinge-Clay and Stroud then examined sequences of many PKS modules and found a KR structural domain upstream of nearly all assigned KR catalytic domains. The KR structural domain is less well-conserved than the KR catalytic domain but is easily recognized by an amino-terminal peptide that makes critical contacts with the catalytic domain. The newly assigned KR structural domain is nearly 200 amino acids in length, and thus, a large region of blank “linker” sequence can now be assigned across the family of type I PKS modules. One of the most surprising findings is that, in PKS modules containing both ER and KR domains (erythromycin PKS module 1 lacks an ER domain), the ER domain is between KR structural and catalytic domains in the polypeptide sequence.
Figure 2. FAS and PKS multifunctional protein structures. a) Crystal structure of porcine FAS (6, PDB code 2CF2). Individual domains are colored separately and linked sequentially as shown in Figure 1 (KS-AT-DH-ER-KR). Dotted lines indicate the locations of unassigned electron density in the FAS map. b) Crystal structure of the KS-AT di-domain from module 5 of the erythromycin PKS (4, PDB code 2HG4). The sequence of domains along the polypeptide is N-dock-KS-link-AT-“stickytape”. The overall position and orientation of KS (blue) and AT (green) domains are strikingly similar in the PKS module and in the type I FAS shown in panel a. c) FAS structure with missing domains modeled from PKS structures. The KS-AT linker (yellow) and the “sticky-tape” (red) are from the structure of the KS-AT di-domain structure shown in panel b. The FAS KR (magenta) is overlaid with the KR catalytic domain (dark blue) from module 1 of the erythromycin PKS (5, PDB code 2FR1); the partner PKS KR structural domain is shown in light blue. VOL.1 NO.8 • 505–509 • 2006
507
The emerging model of catalytic domains is also consistent with the emerging model of a common architecture for PKS modules and the type I FAS.
Each of the FAS and KR crystal structures provides important clues about missing regions of the other. One of the regions of unassigned electron density in the FAS map is adjacent to the KR catalytic domain and seems likely to be a KR structural domain (5), given the superposition of the PKS and FAS KR catalytic domains (Figure 2, panel c). Indeed, the FAS sequence includes a large unassigned “linker” region upstream of the ER domain, in an analagous position to the newly assigned KR structural domain of PKS modules. If the type I FAS and the PKS module have a common organization of KR structural and catalytic domains, then the dimeric organization of DH and ER domains is also likely to be common. The connection of PKS and FAS architecture is even stronger in the third new crystal structure, by Khosla and colleagues (4), of the KS-AT region from module 5 of the erythromycin PKS, the first view of a modular PKS di-domain. The di-domain is an extended dimer with KS domains at the center joined to peripheral AT domains by a linker domain of ⬃100 amino acids. The structure also includes 30 amino acids of the linker sequence following the AT domain. This extended C-terminal peptide acts as a crucial “sticky-tape” in the overall structure, making extensive hydrophobic contacts as it wraps around the linker and KS domains ending near the dimer axis. One of the most exciting aspects of this structure is its striking similarity to the lower half of the type I FAS structure in overall size and shape of the dimer and in the positions of the KS and AT domains (Figure 2). These structural similarities, as well as sequence similarities in the KS-AT linker domains and in the stickytape peptides of PKS modules and type I FASs, imply a similar architecture for the two multifunctional proteins (4). In accord with this idea, unassigned electron density in the FAS map lies between KS and AT domains in exactly the same position as the linker domain in the PKS. Likewise, the sticky-tape peptide, mapped onto the FAS structure, 508
VOL.1 NO.8 • 505–509 • 2006
would end exactly at the waist of the structure where the lower half (KS-AT) connects to the upper half (DH-ER-KR-ACP-TE). Thus, it appears that PKS modules may also have DH-ER-KR-ACP-(TE) upper and KS-AT lower halves joined at a waist. With the significant new insights gained in these recent studies, what structural or functional questions remain to be clarified in the type I FAS and PKS systems? Moreover, what opportunities for practical applications exist given the new information gained from the combined information of these crystal structures? Now that the close structural and functional similarities between type I mammalian FAS and bacterial modular PKSs are so evident, a mystery remains. What is the basis for the protein biochemical metamorphosis of a primordial FAS (that operates as an iterative system to generate an 18-carbon saturated fatty acid) to a multimodular PKS (where an individual catalytic domain functions only once in a sequential series of steps to generate a complex polyketide molecule)? The most salient unique element of modular PKSs is the N-terminal and C-terminal docking domains (Figure 3) that mediate specific module–module interactions. This feature enables cognate pairs of PKS proteins to transfer sequentially an individual linear acyl chain elongation intermediate from one module to another in a process called channeling (Figure 1, panel b). The molecular details of this fascinating process remain obscure, but again, the new structures provide a coherent view. The PKS di-domain structure includes a portion of the N-terminal docking domain, which is its “ticket” to molecular recognition and association with the preceding PKS module. The coiled-coil helices at the dimer axis are consistent with an NMR structure of the isolated docking domains (13), demonstrating that the basis for successful docking is formation of a four-helix bundle with the C-terminus of the previous module. The emerging model of docking domains is also consistent with SHERMAN AND SMITH
Figure 3. Model of docked PKS modules. A hypothetical PKS module nⴙ1 is assembled from the PKS and FAS structures shown in Figure 2, with catalytic domains colored as in Figure 1. The terminal module nⴙ1 also includes an ACP domain from the solution structure of the actinorhodin PKS ACP (18, PDB code 2AF8) and a terminal TE domain from module 6 of the pikromycin PKS (15, PDB code 1HFK). The N-terminal docking domain (brown) of module nⴙ1 associates with the C-terminal docking domain (gold and blue) of module n, as seen in the solution structure of fused docking domains from modules 2 and 3 of the erythromycin PKS (13, PDB codes 1PZQ and 1PZR). Flexible connections between domains are shown as dotted lines. The lower half of module n is shown in outline form.
the emerging model of a common architecture for PKS modules and the type I FAS. The docking domain coiled-coil extends from the di-domain on the opposite side from the sticky-tape linker peptide. This corresponds to the bottom of the lower half of an intact module. The complementary docking www.acschemicalbiology.org
Point of
VIEW domain at the C-terminus of the preceding module would presumably extend from the ACP domains of its upper half (Figure 3). The remarkable insights provided by these new structural studies were accomplished with native proteins in the absence of natural substrates or substrate mimics. Further understanding, particularly details about modular PKS protein–protein interactions, substrate channeling, dynamics, and catalysis, will require cocrystal structures with appropriate molecular probes or affinity labels, in conjunction with protein NMR analysis. Several new cocrystal structures of the pikromycin PKS terminal TE domain involving affinity labels based on natural substrates have recently demonstrated the power of this approach (14, 15). Ultimately, understanding the basis for molecular recognition and substrate channeling in modular PKSs will be of wideranging practical importance for scientists working with these metabolic systems. Although progress has been made toward empirical engineering of PKSs that are functional and able to generate new compounds, these examples are limited to two or three modules (specifically, heterologous modules or catalytic domains derived from phylogenetically related bacteria) that generate tri- or tetraketide metabolites in modest yields (16). When these approaches enable de novo design of polyketide pathways that are versatile, are scalable, and provide efficient access to structurally diverse products, the ability to create new natural product chemical entities will go sky high (17). Acknowledgment: We thank David L. Akey for assistance in building the structural models displayed in Figures 2 and 3. Work on modular PKSs in the authors’ laboratories is supported by National Institutes of Health grants GM076477 (D.H.S.) and DK042303 (J.L.S.).
REFERENCES 1. Nguyen, M., and Chung, E. P. (2005) Telithromycin: the first ketolide antimicrobial, Clin. Ther. 27, 1144–1163.
www.acschemicalbiology.org
2. Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J., and Leadlay, P. F. (1990) An unusually large multifunctional polypeptide in the erythromycinproducing polyketide synthase of Saccharopolyspora erythraea, Nature 348, 176–178. 3. Donadio, S., Staver, M. J., McAlpine, J. B., Swanson, S. J., and Katz, L. (1991) Modular organization of genes required for complex polyketide biosynthesis, Science 252, 675–679. 4. Tang, Y., Kim, C. Y., Mathews, I. I., Cane, D. E., and Khosla, C. (2006) The 2.7-Å crystal structure of a 194-kDa homodimeric fragment of the 6-deoxyerythronolide B synthase, Proc. Natl. Acad. Sci. U.S.A. 103, 11124–11129. 5. Keatinge-Clay, A. T., and Stroud, R. M. (2006) The structure of a ketoreductase determines the organization of the beta-carbon processing enzymes of modular polyketide synthases, Structure 14, 737–748. 6. Maier, T., Jenni, S., and Ban, N. (2006) Architecture of mammalian fatty acid synthase at 4.5 A resolution, Science 311, 1258–1262. 7. Jenni, S., Leibundgut, M., Maier, T., and Ban, N. (2006) Architecture of a fungal fatty acid synthase at 5 Å resolution, Science 311, 1263–1267. 8. Collie, J. (1907) Derivatives of the multiple keten group, J. Chem. Soc. 91, 1806–1813. 9. Hopwood, D. A., and Sherman, D. H. (1990) Molecular genetics of polyketides and its comparison to fatty acid biosynthesis, Annu. Rev. Genet. 24, 37–66. 10. Aldrich, C. C., Venkatraman, L., Sherman, D. H., and Fecik, R. A. (2005) Chemoenzymatic synthesis of the polyketide macrolactone 10-deoxymethynolide, J. Am. Chem. Soc. 127, 8910–8911. 11. Chang, Z., Sitachitta, N., Rossi, J. V., Roberts, M. A., Flatt, P. M., Jia, J., Sherman, D. H., and Gerwick, W. H. (2004) Biosynthetic pathway and gene cluster analysis of curacin A, an antitubulin natural product from the tropical marine cyanobacterium Lyngbya majuscula, J. Nat. Prod. 67, 1356–1367. 12. Edwards, D. J., Marquez, B. L., Nogle, L. M., McPhail, K., Goeger, D. E., Roberts, M. A., and Gerwick, W. H. (2004) Structure and biosynthesis of the jamaicamides, new mixed polyketide-peptide neurotoxins from the marine cyanobacterium Lyngbya majuscula, Chem. Biol. 11, 817–833. 13. Broadhurst, R. W., Nietlispach, D., Wheatcroft, M. P., Leadlay, P. F., and Weissman, K. J. (2003) The structure of docking domains in modular polyketide synthases, Chem. Biol. 10, 723–731. 14. Giraldes, J. W., Akey, D. L., Kittendorf, J. D., Sherman, D. H., Smith, J. L., and Fecik, R. A. (2006) Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels, Nat. Chem. Biol. 2, 531–536. 15. Akey, D. L., Kittendorf, J. D., Giraldes, J. W., Fecik, R. A., Sherman, D. H., and Smith, J. L. (2006) Structural basis for macrolactonization by the pikromycin thioesterase, Nat. Chem. Biol. 2, 537–542. 16. Menzella, H. G., Reid, R., Carney, J. R., Chandran, S. S., Reisinger, S. J., Patel, K. G., Hopwood, D. A., and Santi, D. V. (2005) Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes, Nat. Biotechnol. 23, 1171–1176.
17. Gonzalez-Lergier, J., Broadbelt, L. J., and Hatzimanikatis, V. (2005) Theoretical considerations and computational analysis of the complexity in polyketide synthesis pathways, J. Am. Chem. Soc. 127, 9930–9938. 18. Crump, M. P., Crosby, J., Dempsey, C. E., Parkinson, J. A., Murray, M., Hopwood, D. A., and Simpson, T. J. (1997) Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2), Biochemistry 36, 6000–6008.
VOL.1 NO.8 • 505–509 • 2006
509
