Reaction mechanism of iterative minimal polyketide synthases (PKS) Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life 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

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

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

CoA S Coenzym A (CoA) Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life 4-phosphopantetheineAdeninRibo-3’-phosphat = 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. Next

CoA S AT ACP KS TE SH Loading of a starter unitStarter unit (acetyl-CoA) Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S AT ACP KS TE SH CoA S Loading of a starter unit Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S CoA SH AT ACP KS TE S SH Loading of a starter unit SH Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S SH CoA SH AT ACP KS TE S SH Loading of a starter unit SH Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

SH CoA SH AT ACP KS TE SH S Loading of a starter unit 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. Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

Activation of extender units CoA S + CO 2 Acetyl-CoA Carboxylase CoA S The CO 2 originates from a HCO 3 - bond to biotin in the enzyme Acetyl-CoA Malonyl-CoA Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

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

SSH CoA S AT ACP KS TE SH S Loading of a extender unit SH Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S S CoA SH AT ACP KS TE SH S Loading of a extender unit Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

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

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

SH CoA SH AT ACP KS TE S SH S Preparing for a second round SH Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

SSH AT ACP KS TE S SH S CoA S Loading of the 2 nd extender unit SH Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

KS TE SH S CoA SH S S AT ACP SH Loading of the 2 nd extender unit Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

KS TE SH S CoA SH AT ACP S SH 2 nd condensation Decarboxylation Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S KS TE SH S CoA SH AT ACP S SH Release from the enzyme S Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life 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. Next

S KS TE SH S CoA SH AT ACP S SH Release from the enzyme SH Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S KS TE S CoA SH AT ACP S SH Release from the enzyme SH Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S KS TE S CoA SH AT ACP S SH Release from the enzyme SH Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S KS TE S CoA SH AT ACP S SH Release from the enzyme SH HO Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

S KS TE S CoA SH AT ACP S SH Release from the enzyme SH Starter unit 1 st extender unit 2 nd extender unit Rasmus J.N. Frandsen 2007 HO Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life Next

Release from the enzyme Starter unit 1 st extender unit 2 nd extender unit Rasmus J.N. Frandsen 2007 HO Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life 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. Next

Where does the diversity originate from? Rasmus J.N. Frandsen 2007Rasmus J.N. Frandsen 2007 University of Copenhagen, Faculty of Life 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:  -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