title: Domain analysis of the molecular recognition features of aromatic polyketide synthase subunits.
abstract: Bacterial aromatic polyketide synthases (PKSs) are a family of homologous multienzyme assemblies that catalyze the biosynthesis of numerous polyfunctional aromatic natural products. In the absence of direct insights into their structures, the use of gene fusions can be a powerful tool for understanding the structural basis for their properties. A series of truncated and hybrid proteins were constructed and analyzed within a family of PKS subunits, designated aromatases/cyclases (ARO/CYCs). When expressed alone, neither the N-terminal nor the C-terminal domain of the actinorhodin (act) or the griseusin (gris) ARO/CYC exhibited substantial aromatase activity. However, in the presence of each other, the half proteins were active. Furthermore, analysis of a set of hybrid proteins derived from the act and gris ARO/CYCs allowed us to localize the chain length dependence of this aromatase activity to their N-terminal domains. Unexpectedly, however, when the C-terminal domain of the gris ARO/CYC was expressed in a context where aromatase activity was absent, it could modulate the chain length specificity of the tetracenomycin (tcm) minimal PKS, leading to the formation of a novel 18-carbon product in addition to the expected 20-carbon one. It was also found that monodomain ARO/CYCs such as tcmN cannot substitute for the the N-terminal domain of didomain ARO/CYCs, even though they exhibit high sequence similarity with the N-terminal domain. Together, these results illustrate the utility of protein engineering approaches for dissecting the structure-function relationships of PKS subunits and for the generation of mutant alleles with novel biosynthetic properties.
authors.affiliation: Department of Chemical Engineering, Stanford University, Stanford, California 94305-5025, USA.
authors.forename: R J
authors.initials: RJ
authors.lastname: Zawada
authors.forename: C
authors.initials: C
authors.lastname: Khosla
mesh_terms: D000595:Amino Acid Sequence; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013329:Structure-Activity Relationship
chemical_list: D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins