Structural and computational studies of bacterial acyl carrier proteins.

摘要

Acyl carrier protein (ACP) is the essential cofactor protein in fatty acid biosynthesis that covalently binds all intermediates during the synthetic process. In contrast to mammalian, type I fatty acid synthase (FAS) systems, where ACP is part of a larger multienzyme complex, in dissociated, type II bacterial systems ACP exists as a 9 kDa, overall highly acidic, individual protein. This thesis presents molecular dynamics (MD) simulations of saturated acyl chains bound to Escherichia coli acyl-ACP, as well as fatty acid synthesis intermediates bound to this protein. Further, the NMR solution structure of Vibrio harveyi ACP A75H and its 15N NMR backbone dynamics are presented. Our studies provide a detailed set of MD simulations on ACP for the first time, revealing novel information on the intricacies of acyl chain accommodation by ACP. The simulations indicate that the acyl groups are bound in two sub-cavities within the protein and that the shorter acyl chains, up to eight carbon groups in length, are fully solvent shielded. Longer attachments are increasingly solvent exposed and confer higher flexibility to portions of ACP. We show that the beta-ketoacyl-, beta-hydroxyacyl-, and trans-2-enoyl-intermediates display different modes of association with ACP. This yields a rationale for how different acyl-ACP forms are distinguished by partner enzymes in the FAS system. The solution structure of V. harveyi ACP A75H reveals that His75, a unique stabilizing residue of this protein, is located adjacent to a large anionic domain of ACP. NMR pH titration studies suggest that the anionic domain is the least stable region of ACP and that the positive charge of His75 reduces the destabilizing charge repulsion in this domain. His75 also has a profound effect on the calcium and magnesium binding ability of V. harveyi ACP through its interaction with the same anionic portion, which also encompasses the divalent cation binding sites. The backbone dynamics illustrate that the region involved in enzyme interactions possesses large flexibilities and that the helix containing His75 is remarkably rigid. Furthermore, the order parameters reveal a spatial organization to the flexibility of ACP.