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 Domains in a minimal polyketide syntase AT domain = Acyltransferase AT SH SH Acyl Carrier protein (ACP) ACP b-ketoacyl synthase (KS) KS SH Thioesterase (TE) TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Domains in a minimal polyketide syntase AT domain = Acyltransferase AT SH SH Acyl Carrier protein (ACP) ACP b-ketoacyl synthase (KS) KS SH Thioesterase (TE) TE SH O S er H2 C Prosthetic group: 4-phosphopantetheine (PPT). A flexible group that can transfer the starter and extender units internally in the enzyme. O P H 3C CH3 O O SH N H OH ACP O N H OH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences 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. NH2 N N O N N H 2C O O P O P H 3C CH3 O O SH O OH O N H OH N H OH OH O O P OH OH Adenin Ribo-3’-phosphat 4-phosphopantetheine = CoA S Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences O Loading of a starter unit CoA AT S C CH3 Starter unit (acetyl-CoA) SH SH ACP KS SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences O Loading of a starter unit CoA AT S C CH3 S SH SH ACP KS SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of a starter unit CoA SH O AT SH S C S CH3 ACP KS SH TE SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of a starter unit CoA AT SH SH ACP O SH S KS S SH TE SH C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of a starter unit CoA AT SH SH ACP SH O KS S TE SH C CH3 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 Activation of extender units O Acetyl-CoA CoA S C CH3 The CO2 originates from a HCO3- bond to biotin in the enzyme Malonyl-CoA + CO2 Acetyl-CoA Carboxylase CoA S O O C C OH C H2 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of a extender unit CoA S O O C C OH C H2 AT Extender unit (malonyl-CoA) SH ACP SH O KS S TE SH C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of a extender unit CoA S SH O O C C OH C H2 AT S SH ACP SH O KS S TE SH C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of a extender unit CoA AT SH S SH O O C C OH C H2 S SH ACP O KS S TE SH C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Ready for condensation Decarboxylation of the extender unit (malonyl) provides the energy/electron for the condensation CoA AT SH SH ACP S O O C C C H2 O KS S TE SH C O- CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Condensation Decarboxylation of the extender unit (malonyl) provides the energy/electorne for the codensation CoA AT SH SH ACP S O OO C CC C H2 O KS S SH TE SH C OO- CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Preparing for a second round CoA AT SH SH O ACP S SH O C C H2 KS SH S TE SH C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of the 2nd extender unit CoA S SH O O C C OH C H2 AT SH ACP S SH O KS S O C C H2 TE C CH3 SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Loading of the 2nd extender unit CoA AT SH S SH O O C C OH C H2 S SH ACP O KS S O C C H2 TE C CH3 SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences 2nd condensation CoA SH Decarboxylation AT SH ACP S O O C C CH2 O KS S SH O C C H2 TE O C CH3 SH Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Release from the enzyme 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. CoA AT SH SH O ACP S O C CH2 KS S SH TE S SH O C C H2 C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Release from the enzyme CoA AT SH SH O ACP S SH O C CH2 KS S SH TE S SH O C C H2 C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Release from the enzyme CoA AT SH SH ACP KS S SH S SH O TE S O C CH2 O C C H2 C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Release from the enzyme CoA AT SH SH ACP KS S SH S SH O TE S O C CH2 O C C H2 C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Release from the enzyme CoA AT SH SH ACP KS S SH S SH O TE S SH O C CH2 HO O C C H2 C CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences Release from the enzyme CoA AT SH SH ACP S SH KS S SH TE S SH O HO 2nd O C extender unit CH2 1st extender unit O C C H2 C Starter unit CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences 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. O HO O C CH2 C O C C H2 CH3 Next Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences 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: O b-ketoacyl reductase (KR) C OH CH H 2C H 2C OH Dehydratase (DH) CH CH HC H 2C Enoyl reductase (ER) CH Methyltransferase (MET) CH2 HC H 2C OH O CH H 2C CH3 CH H 2C Cyclases (Cyc) – fold the polyketide chain into an aromatic or macrocyclic compound + alternative extender units different from malonyl-CoA Rasmus J.N. Frandsen 2007 (raf@life.ku.dk) University of Copenhagen, Faculty of Life Sciences END