The long-term objective is to elucidate the structure, mechanism of action and basis for specificity of the animal fatty acid synthase (FAS), a multifunctional polypeptide that contains all of the enzymes required for biosynthesis of fatty acids from malonyl-CoA. Evidence has been obtained, by mutant complementation analysis that challenges the classical antiparallel subunit model for the FAS homodimer. An alternative model is proposed in which the two amino-terminal domains, beta-ketoacyl synthase, and malonyl/acetyltransferase, cooperate with the penultimate carboxy-terminal ACP domain of either subunit, whereas catalysis of the beta-carbon processing and chain-terminating reactions occurs exclusively within one subunit. A novel strategy that permits engineering of FAS heterodimers independently modified in each subunit will be exploited to test and refine the new model, as follows. The ability of dibromopropanone to form cross-links between the phosphopantetheine and the beta-ketoacyl synthase nucleophile both inter- and intra-subunit, as predicted by the new model, will be evaluated. The ability of a mutated subunit, functionally compromised in all seven domains, to provide the structural scaffold required for supporting fatty acid synthesis by a wild-type subunit partner, as predicted by the new model, will be tested. Panels of heterodimeric FAS mutants also will be utilized to assess the relative contributions of the alternative mechanisms for substrate delivery and condensation. The role of interdomain linkers in facilitating dynamic interactions between functional domains will be evaluated by the introduction of new protease sites within the linkers, and the effects of cleavage on structure and function of the FASs will be determined. The possible role of both catalytic and structural domains in maintaining subunit interactions in the dimer will be explored by identifying sequence elements that engage in hetero- and/or homodimeric interactions. A structural context for the emerging functional model will be sought, primarily using a combination of single particle and two-dimensional crystallographic analysis by electron microscopy. A novel strategy is described that permits imaging of the FAS in different conformational states. A detailed analysis of the catalytic mechanisms of key functional domains will be pursued and elucidation of the three-dimensional structure of these individual domains will be sought by x-ray crystallography. These studies will continue to provide a model for understanding the domain interactions and catalytic mechanisms operative in modular polyketide synthases that are responsible for the biosynthesis of clinically important antibiotics, anticancer and immunosuppressive agents.
Showing the most recent 10 out of 50 publications
