Dr. Young-OK You, George Mason University
Host: Dr. Songon An
Function, mechanism and regulation of a mediator domain of trans-AT polyketide synthase
Type I modular polyketide synthase (PKS) is a group of multi-enzyme complexes which produce various macrolides such as erythromycin, FK506 (Tacrolimus), rifamycin and rapamycin. The modular type I PKSs are characterized by the linear assembly of modules containing functional domains analogous to the fatty acid synthase. The relatively new class of PKS is called “trans-acyltransferase (AT)- PKS”. A recent computational analysis of sequenced bacterial genomes revealed that more than one-third of bacterial modular PKS belongs to trans-AT PKSs, implying that trans-AT PKS are a major class of natural products biosynthases. The trans-AT PKS contain novel enzyme functions, atypical modular organizations including split modules, non-elongation modules, and unusual domain orders.
One of the frequently observed novel enzyme is the condensation-incompetent ketosynthase domain (KS0). KS0 is found in associated with various enzymes, such as dehydratase, enoylreductase, O-methyltransferase, thioesterase, active ketosynthase, and at the interface of PKS-NRPS. It cannot synthesize carbon-carbon bond formation as normal KS does, but it has been demonstrated as essential enzyme. The past few years of study have shown this enzyme contains a gate-keeping function as well as intermodular translocation function. Our recent study of difficidine biosynthase KS05 revealed that it performs intermodular translocation aiding the associated dehydratase domain activity. The mediating functions are also observed in other PKS synthases similarly aiding other enzyme activities. The modification of associated acyl carrier protein (ACP) is also regulated by KS0. Our enzyme kinetics revealed that KS0 is an allosteric inhibitor of AT. In addition, our high-resolution mass spectrometry data showed that the modification of associated ACP is mainly dominated and regulated by KS0.
The promise and expectation of PKS engineering is to generate novel macrolides including novel antibiotics, non-immunosuppressant FKBP inhibitors. However, the specificity between the protein components need to be overcome. Our research showed the mediating activity of KS0 is essential in circumvent the specificity. We expect that KS0 can be utilized as a tool of synthetic biology and protein engineering.