| Modular polyketide synthases (PKSs), such as 6-deoxyerythronolide B synthase (DEBS), represent examples of multifunctional enzymes responsible for the biosynthesis of structurally complex molecules from simple precursors. DEBS assembles six three-carbon precursors into the macrolide aglycon of the antibiotic, erythromycin. The polyketide product reflects the architecture of the enzymatic system, as DEBS comprises six “modules”, which are each responsible for the addition and processing of one three-carbon extender unit. Thus, the polyketide product reflects the sequence of the modules in the PKS. The modular organization of DEBS has attracted interest due to the potential for engineering enzymes to produce novel polyketides. However, the ability to re-order entire modules of PKSs has met with little success due to the lack of understanding of the communication between modules.; Modules exhibit remarkable selectivity by processing a specific intermediate coming from a specific module. Whether modules rely on recognition of the substrate or the preceding module was a question that had received little insight. In this thesis, the contribution of recognition mediated by protein-protein interactions was investigated.; It was revealed that, surprisingly, short, non-conserved “linker” regions flanking the catalytic domains of the modules play an important role. In intrapolypeptide transfer, changes of two amino acids in the linker eliminated detectable activity while their maintenance allowed successful transfer to heterologous modules. For interpolypeptide transfer, the linkers were found to function in pairs, where linkers on the C- and N-termini of consecutive modules (but on different proteins) mediated transfer. Not only were these interpolypeptide linkers shown to be portable to different module pairings, but they were also shown to facilitate transfer between non-consecutive modules. Because of their importance in intermodular communication, replacement of either terminal linker of a natural pair greatly reduced transfer. Preliminary structural studies of synthetic peptide mimics of the linkers have also suggested a possible coiled-coil framework for these interactions.; These advances will help to unlock the obvious potential of PKSs to generate a diversity of new products. |
