An antibiotic factory caught in action


The synthesis of aromatic polyketides, such as actinorhodin, tetracycline and doxorubicin, begins with the formation of a polyketide chain. In type II polyketide synthases (PKSs), chains are polymerized by the heterodimeric ketosynthase–chain length factor (KS-CLF). Here we present the 2.0-Å structure of the actinorhodin KS-CLF, which shows polyketides being elongated inside an amphipathic tunnel 17 Å in length at the heterodimer interface. The structure resolves many of the questions about the roles of KS and CLF. Although CLF regulates chain length, it does not have an active site; KS must catalyze both chain initiation and elongation. We provide evidence that the first cyclization of the polyketide occurs within the KS-CLF tunnel. The mechanistic details of this central PKS polymerase could guide biosynthetic chemists in designing new pharmaceuticals and polymers.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Octaketide production by the actinorhodin minimal PKS.
Figure 2: KS-CLF complementarity.
Figure 3: The polyketide tunnel.
Figure 4: Polyketide intermediates.

Accession codes


Protein Data Bank


  1. 1

    Hopwood, D.A. Genetic contributions to understanding polyketide synthases. Chem. Rev. 97, 2465–2498 (1997).

  2. 2

    Rawlings, B.J. Biosynthesis of polyketides (other than actinomycete macrolides). Nat. Prod. Rep. 16, 425–484 (1999).

  3. 3

    Dreier, J., Shah, A.N. & Khosla, C. Kinetic analysis of the actinorhodin aromatic polyketide synthase. J. Biol. Chem. 274, 25108–25112 (1999).

  4. 4

    Carreras, C.W. & Khosla, C. Purification and in vitro reconstitution of the essential protein components of an aromatic polyketide synthase. Biochemistry 37, 2084–2088 (1998).

  5. 5

    McDaniel, R., Ebert-Khosla, S., Hopwood, D.A. & Khosla, C. Rational design of aromatic polyketide natural products by recombinant assembly of enzymatic subunits. Nature 375, 549–554 (1995).

  6. 6

    Tang, Y., Tsai, S.C. & Khosla, C. Polyketide chain length control by chain length factor. J. Am. Chem. Soc. 125, 12708–12709 (2003).

  7. 7

    He, M., Varoglu, M. & Sherman, D.H. Structural modeling and site-directed mutagenesis of the actinorhodin β-ketoacyl-acyl carrier protein synthase. J. Bacteriol. 182, 2619–2623 (2000).

  8. 8

    Mathieu, M. et al. The 2.8 Å crystal structure of peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: a five-layered αβαβα structure constructed from two core domains of identical topology. Structure 2, 797–808 (1994).

  9. 9

    Huang, W. et al. Crystal structure of β-ketoacyl-acyl carrier protein synthase II from E. coli reveals the molecular architecture of condensing enzymes. EMBO J. 17, 1183–1191 (1998).

  10. 10

    Sherman, D.H., Kim, E.S., Bibb, M.J. & Hopwood, D.A. Functional replacement of genes for individual polyketide synthase components in Streptomyces coelicolor A3(2) by heterologous genes from a different polyketide pathway. J. Bacteriol. 174, 6184–6190 (1992).

  11. 11

    Bisang, C. et al. A chain initiation factor common to both modular and aromatic polyketide synthases. Nature 401, 502–505 (1999).

  12. 12

    Witkowski, A., Joshi, A.K., Lindqvist, Y. & Smith, S. Conversion of a β-ketoacyl synthase to a malonyl decarboxylase by replacement of the active-site cysteine with glutamine. Biochemistry 38, 11643–11650 (1999).

  13. 13

    Fernandez-Moreno, M.A., Martinez, E., Boto L., Hopwood, D.A. & Malpartida, F. Nucleotide sequence and deduced functions of a set of cotranscribed genes of Streptomyces coelicolor A3(2) including the polyketide synthase for the antibiotic actinorhodin. J. Biol. Chem. 267, 19278–19290 (1992).

  14. 14

    Harris, T.M. & Harris, C.M. Biomimetic syntheses of aromatic polyketide metabolites. Pure Appl. Chem. 58, 283–294 (1986).

  15. 15

    Dreier, J. & Khosla, C. Mechanistic analysis of a type II polyketide synthase. Role of conserved residues in the β-ketoacyl synthase–chain length factor heterodimer. Biochemistry 39, 2088–2095 (2000).

  16. 16

    Tang, Y., Lee, T.S. & Khosla, C. Engineered biosynthesis of regioselectively modified aromatic polyketides using bimodular polyketide synthases. PLoS Biol. 2, 227–238 (2004).

  17. 17

    Ferrer, J.L., Jez, J.M., Bowman, M.E., Dixon, R.A. & Noel, J.P. Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis. Nat. Struct. Biol. 6, 775–784 (1999).

  18. 18

    McDaniel, R., Ebert-Khosla, S., Hopwood, D.A. & Khosla, C. Engineered biosynthesis of novel polyketides: manipulation and analysis of an aromatic polyketide synthase with unproven catalytic specificities. J. Am. Chem. Soc. 115, 11671–11675 (1993).

  19. 19

    Summers, R.G., Wendt-Pienkowski, E., Motamedi, H., & Hutchinson, C.R. The tcmVI region of the tetracenomycin C biosynthetic gene cluster of Streptomyces glaucescens encodes the tetracenomycin F1 monooxygenase, tetracenomycin F2 cyclase, and, most likely, a second cyclase. J. Bacteriol. 175, 7571–7580 (1993).

  20. 20

    Kramer, P.J. et al. Rational design and engineered biosynthesis of a novel 18-carbon aromatic polyketide. J. Am. Chem. Soc. 119, 635–639 (1997).

  21. 21

    Keatinge-Clay, A.T. et al. Catalysis, specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase. Structure (Camb.) 11, 147–154 (2003).

  22. 22

    Crump, M.P. et al. Solution structure of the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2). Biochemistry 36, 6000–6008 (1997).

  23. 23

    Sciara, G. et al. The structure of ActVA-Orf6, a novel type of monooxygenase involved in actinorhodin biosynthesis. EMBO J. 22, 205–215 (2003).

  24. 24

    Pan, H. et al. Crystal structure of the priming β-ketosynthase from the R1128 polyketide biosynthetic pathway. Structure (Camb.) 10, 1559–1568 (2002).

  25. 25

    McDaniel, R., Ebert-Khosla, S., Hopwood, D.A. & Khosla, C. Engineered biosynthesis of novel polyketides. Science 262, 1546–1550 (1993).

  26. 26

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  27. 27

    Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

  28. 28

    Biemann, K. Appendix 5. Nomenclature for peptide fragment ions (positive ions). Methods Enzymol. 193, 886–887 (1990).

Download references


We thank Y. Tang and S. Kobayashi for valuable conversations about KS-CLF biochemistry and help in transforming into and purifying from S. coelicolor. Research was supported by US National Institutes of Health (NIH) Cancer Institute grants CA 63081 (R.M.S.) and CA 77248 (C.K.). A.T.K. also received a fellowship from the Achievement Rewards for College Scientists Foundation. D.A.M. and K.F.M. were supported by US NIH National Center for Research Resources grants RR 01614 and RR 12961 (to the UCSF MS Facility, director A.L. Burlingame).

Author information

Correspondence to Robert M Stroud.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

KS-CLF secondary structure. (PDF 305 kb)

Supplementary Fig. 2

The proposed CLF active site. (PDF 354 kb)

Rights and permissions

Reprints and Permissions

About this article

Further reading