title: Effect of modification of the length and flexibility of the acyl carrier protein-thioesterase interdomain linker on functionality of the animal fatty acid synthase.
abstract: A natural linker of approximately 20 residues connects the acyl carrier protein with the carboxy-terminal thioesterase domain of the animal fatty acid synthase. This study examines the effects of changes in the length and amino acid composition of this linker on catalytic activity, product composition, and segmental motion of the thioesterase domain. Deletion of 10 residues, almost half of the interdomain linker, had no effect on either mobility of the thioesterase domain, estimated from fluorescence polarization of a pyrenebutyl methylphosphono moiety bound covalently to the active site serine residue, or functionality of the fatty acid synthase; further shortening of the linker limited mobility of the thioesterase domain and resulted in reduced fatty acid synthase activity and an increase in product chain length from 16 to 18 and 20 carbon atoms. Surprisingly, however, even when the entire linker region was deleted, the fatty acid synthase retained 28% activity. Lengthening of the linker, by insertion of an unusually long acyl carrier protein-thioesterase linker from a modular polyketide synthase, increased mobility of the thioesterase domain without having any significant effect on catalytic properties of the complex. Interdomain linkers could also be used to tether, to the acyl carrier protein domain of the fatty acid synthase, a thioesterase active toward shorter chain length acyl thioesters generating novel short-chain fatty acid synthases. These studies reveal that although truncation of the interdomain linker partially impacts the ability of the thioesterase domain to terminate growth of the acyl chain, the overall integrity of the fatty acid synthase is quite tolerant to moderate changes in linker length and flexibility. The retention of fatty acid synthesizing activity on deletion of the entire linker region implies that the inherent flexibility of the phosphopantetheine "swinging arm" also contributes significantly to the successful docking of the long-chain acyl moiety in the thioesterase active site.
authors.affiliation: Children's Hospital Oakland Research Institute, 5700 Martin Luther King Jr. Way, Oakland, California 94609, USA.
authors.forename: Anil K
authors.initials: AK
authors.lastname: Joshi
authors.forename: Andrzej
authors.initials: A
authors.lastname: Witkowski
authors.forename: Harvey A
authors.initials: HA
authors.lastname: Berman
authors.forename: Lei
authors.initials: L
authors.lastname: Zhang
authors.forename: Stuart
authors.initials: S
authors.lastname: Smith
mesh_terms: D000213:Acyl Carrier Protein / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D064429:Fatty Acid Synthases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010170:Palmitoyl-CoA Hydrolase / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D017384:Sequence Deletion; D013379:Substrate Specificity / Q000235:genetics; D013816:Thermodynamics; D013869:Thiolester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000213:Acyl Carrier Protein; D010446:Peptide Fragments; D064429:Fatty Acid Synthases; D013869:Thiolester Hydrolases; C022738:thioesterase II; C018748:oleoyl-(acyl-carrier-protein) hydrolase; D010170:Palmitoyl-CoA Hydrolase