| Daunorubicin (DNR) and its C-14 hydroxylated derivative doxorubicin (DXR) are the most widely used anthracyclines as anti-tumour agents. DNR and DXR are produced by the soil bacteria Streptomyces peucetius through a biosynthetic pathway that employs a type II polyketide synthase (PKS). Type II PKSs consist of several discrete, monofunctional proteins that form a dissociable complex. Studies on enzyme complex formation and substrate channelling are essential for a better understanding of metabolism and could lead to the generation of novel compounds by 'combinatorial' biosynthesis. The 21-carbon atom aromatic polyketide, aklanonic acid, is the first enzyme-free intermediate in the DNR/DXR biosynthetic pathway. The DNR/DXR PKS is composed of eight proteins: DpsABCDG form the 'minimal' PKS, responsible for the condensation reactions between the propionate starter unit and nine malonate extender units, whereas DpsEFY catalyse successive modifications of the carbon backbone such as reduction, aromatisation and cyclisation. This type II PKS is intriguing since it contains a type III ketosynthase (DpsC) that selects the starter unit and a putative malonyl/acyltransferase (DpsD) whose role seems obscure. The network of protein interactions within this complex have been investigated using a yeast two-hybrid system (GAL4), affinity chromatography (Tandem Affinity Purification) and computer-aided protein docking simulations (Hex 4.5 software). The results have led to the proposal of a head-to-tail arrangement for the 'minimal' PKS suggested by the interactions established by DpsA, DpsB and DpsD. A putative role for DpsD is suggested as physical inhibitor of the incorporation of acetate in the priming reaction allowing the choice of propionate. Also, a structural role for the cyclase DpsY is proposed perhaps to maintain the overall structural integrity of the complex. This represents the first study attempting to analyse in vivo protein interactions forming a type II PKS. The purification method has allowed isolation of DpsA and DpsB and in silico docking simulations have produced results consistent with the proposed arrangements of proteins based on yeast two-hybrid assays. |
