Biosynthesis of Doxorubicin - Polyketide Chain Synthesis

Polyketide Chain Synthesis

Doxorubicin is synthesized by a specialized polyketide synthase.

The initial event in DXR synthesis is the selection of the propionyl-CoA starter unit and its decarboxylative addition to a two carbon ketide unit, derived from malonyl-CoA to produce the five carbon B-ketovaleryl ACP. The five carbon diketide is delivered by the ACP to the cysteine sulfhydryl group at the KS active site, by thioester exchange, and the ACP is released from the chain. The free ACP picks up another malonate group from malonyl-CoA, also by thioester exchange, with release of the CoA.

The ACP brings the new malonate to the active site of the KS where is it decarboxylated, possibly with the help of the CLF subunit, and joined to produce a 7 carbon triketide, now anchored to the ACP (see top of Figure 1). Again the ACP hands the chain off to the KS subunit and the process is repeated iteratively until the decaketide is completed.

In most Type II systems the initiating event is delivery by ACP of an acetate unit, derived from acetyl-CoA, to the active site of the ketosynthase (KS) subunit of the KS/CLF heterodimer. The default mode for Type II PKS systems is the incorporation of acetate as the primer unit, and that holds true for the DXR "minimal PKS". In other words the action of KS/CLF/ACP (Dps A, B and G) from this system will not produce 21-carbon decaketides, but 20-carbon decaketides instead, because acetate is the “preferred” starter. The process of specifying propionate is not completely understood, but it is clear that it depends on an additional protein, Dps C, which may be acting as a ketosynthase or acyltransferase selective for propionyl-CoA, and possibly Dps D makes a contribution.

A dedicated MAT has been found to be dispensable for polyketide production under in vitro conditions. The PKS may "borrow" the MAT from its own fatty acid synthase and this may be the primary way ACP receives its malonate group in DXR biosynthesis. Additionally, there is excellent evidence that "self-malonylation" is an inherent characteristic of Type II ACPs. In summary, a given Type II PKS may provide its own MAT (s), it may borrow one from FAS, or its ACP may “self-malonylate”.

It is unknown whether the same KS/CLF/ACP ternary complex chaperones the growth of a full length polyketide chain through the entire catalytic cycle, or whether the ACP dissociates after each condensation reaction. A 2.0-Å resolution structure of the actinorhodin KS/CLF, which is very similar to the dps KS/CLF, shows polyketides being elongated inside an amphipathic tunnel formed at the interface of the KS and CLF subunits. The tunnel is about 17-Å long and one side has many charged amino acid residues which appear to be stabilizing the carbonyl groups of the chain, while the other side is hydrophobic. This structure explains why both subunits are necessary for chain elongation and how the reactive growing chain is protected from random spontaneous reactions until it is positioned properly for orderly cyclization. The structure also suggests a mechanism for chain length regulation. Amino acid side groups extend into the tunnel and act as "gates". A couple of particularly bulky residues may be impassable by the chain, causing termination. Modifications to tunnel residues based on this structure were able to alter the chain length of the final product. The final condensation causes the polyketide chain to "buckle" allowing an intramolecular attack by the C-12 methylene carbanion, generated by enzyme catalyzed proton removal and stabilized by electrostatic interactions in the tunnel, on the C-7 carbonyl (see 3 in Figure 1). This tunnel aided intramolecular aldol condensation provides the first cyclization when the chain is still in the tunnel. The same C-7/C-12 attack occurs in the biosynthesis of DXR, in a similar fashion.