Reaction mechanism of iterative minimal polyketide synthases (PKS)

1. Reaction mechanism of iterative minimal polyketide synthases (PKS) Polyketide synthases are multidomain enzymes that catalyze the condensation of ketide units (starter unit and extender units) resulting in the formation of polyketides. The reaction is driven by decarboxylation of the extender unit during condensation, which is also known as a Claisen condensation. The motivation for making this animation was that many of our students struggled with understanding how the different substrates and products were moved around inside the PKS, during biosynthesis. The following slides shows the conceptual reaction mechanism and is not correct in chemical terms with respect to the flow of electrons. Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

2. Domains in a minimal polyketide syntase AT AT domain = Acyltransferase SH ACP SH Acyl Carrier protein (ACP) KS SH b-ketoacyl synthase (KS) TE SH Thioesterase (TE) Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

3. Domains in a minimal polyketide syntase AT AT domain = Acyltransferase SH Prosthetic group: 4-phosphopantetheine (PPT). A flexible group that can transfer the starter and extender units internally in the enzyme. ACP SH Acyl Carrier protein (ACP) KS SH b-ketoacyl synthase (KS) TE SH Thioesterase (TE) Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

4. CoA S Coenzym A (CoA) Coenzym A also contains a 4-phosphopantetheine group, similar to that found on the ACP domain of PKSs. The terminal thioester group serves at the attachment point for acetyl and malonyl units. Adenin Ribo-3’-phosphat 4-phosphopantetheine = Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

5. CoA S Loading of a starter unit Starter unit (acetyl-CoA) AT SH ACP SH KS SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

6. CoA S Loading of a starter unit AT S SH ACP SH KS SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

7. Loading of a starter unit CoA SH AT SH S S ACP KS SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

8. Loading of a starter unit CoA SH AT SH ACP SH S KS S SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

9. Loading of a starter unit CoA SH AT SH ACP SH KS S TE SH A starter unit has now been loaded into the KS domain of the PKS and we are ready for loading of the first extender unit. Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

10. CoA CoA S S + CO2 Activation of extender units Acetyl-CoA Acetyl-CoA Carboxylase The CO2 originates from a HCO3- bondto biotin in the enzyme Malonyl-CoA Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

11. Extender unit (malonyl-CoA) Loading of a extender unit CoA S AT SH ACP SH KS S TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

12. Loading of a extender unit CoA S SH AT S SH ACP SH KS S TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

13. Loading of a extender unit CoA SH AT SH S ACP S SH KS S TE SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

14. Ready for condensation CoA SH Decarboxylation of the extender unit (malonyl) provides the energy/electron for the condensation AT SH ACP S KS S TE SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

15. Condensation CoA SH Decarboxylation of the extender unit (malonyl) provides the energy/electorne for the codensation AT SH ACP S 2 KS S SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Rasmus J.N. Frandsen 2007

16. Preparing for a second round CoA SH AT SH ACP S SH KS SH S TE SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

17. Loading of the 2nd extender unit CoA S SH AT S SH ACP S SH KS S TE SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

18. Loading of the 2nd extender unit CoA SH AT SH S ACP S SH KS S TE SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

19. 2nd condensation CoA SH Decarboxylation AT SH ACP S KS S SH TE SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

20. Release from the enzyme CoA SH At this stage the enzyme faces a choice, whether to continue with additional rounds of condensations or to release the polyketide chain from the enzyme. The number of condensation rounds (iterations) that the individual PKSs perform is at present not predictable. One hypothesis is that the size (volume) of the active site in the KS domain could be the deciding factor for total number of iterations possible. AT SH ACP S S KS S SH TE S SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

21. Release from the enzyme CoA SH AT SH ACP SH S KS S SH TE S SH Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

22. Release from the enzyme CoA SH AT SH ACP S SH KS S SH TE S Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

23. Release from the enzyme CoA SH AT SH ACP S SH KS S SH TE S Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

24. Release from the enzyme CoA SH AT SH ACP S SH KS S SH TE SH S HO Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

25. Starter unit 2nd extender unit 1st extender unit Release from the enzyme CoA SH AT SH ACP SH S KS SH S TE S SH HO Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Rasmus J.N. Frandsen 2007

26. Release from the enzyme Note that the formed polyketide chain has polarity. With a methyl (-CH3) group at the ”oldest” end and a carboxyl (-COOH) group at the ”newest” end. HO Starter unit 1st extender unit 2nd extender unit Next Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences

27. Where does the diversity originate from? In addition to the four catalytic domains (AT, ACP, KS and TE) used by the minimal PKS. Other domains can also participate in the biosynthesis: b-ketoacyl reductase (KR) Dehydratase (DH) Enoyl reductase (ER) Methyltransferase (MET) Cyclases (Cyc) – fold the polyketide chain into an aromatic or macrocyclic compound + alternative extender units different from malonyl-CoA END Rasmus J.N. Frandsen 2007 Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences