| Soil bacteria have been waging chemical warfare against each other for billions of years. A major class of antibiotics they secrete are aromatic polyketides. Man has benefitted from these molecules, using them not only as antibiotics, as in the case of tetracycline, but also to fight diseases like cancer, as in the case of doxorubicin. While new polyketides have been created by mixing components from different polyketide synthases, the rational engineering of these enzymes to produce a desired polyketide requires a deeper understanding of their catalysis and specificity. We have structurally and mechanistically analyzed three enzymes involved in the first half of aromatic polyketide synthesis. In the actinorhodin pathway, the minimal PKS---a ketosynthase/chain length factor complex (KS/CLF), an acyltransferase (MAT), and an acyl carrier protein (ACP)---synthesizes an octaketide from eight molecules of malonyl-CoA. In the R1128 PKS, two additional proteins---the priming ketosynthase, ZhuH, and another ACP---synthesize the starter unit that primes the minimal PKS. Our structure of ZhuH bound to a degraded acetyl-CoA indicates why it can accept several small acyl-CoA's and suggests mutations that may further increase the diversity of substrates it accepts. We observed the MAT for the actinorhodin pathway (the Streptomyces coelicolor FabD) bound to an acetate molecule that mimicked the carboxylate of a malonyl group, helping us deduce the structure of the acyl-enzyme intermediate and locate residues that might prevent the transfer of alpha-substituted malonyl groups. KS/CLF was isolated from S. coelicolor during actinorhodin synthesis. Its structure reveals a ∼17 A amphipathic tunnel created at the heterodimer interface. The electron density maps and mass spectrometry data indicate that monoketides and diketides are bound to the reactive cysteine of KS. Gating residues that control chain length were identified. We also identified a water molecule at the midpoint of the polyketide tunnel that may catalyze formation of the first polyketide ring. This information will help biosynthetic chemists to rationally engineer aromatic polyketide synthases capable of producing new pharmaceuticals and polymers. |
