ACYL CARRIER PROTEIN DYNAMICS AND PARTNER ENZYME INTERACTIONS

Abstract

Acyl carrier protein (ACP) is essential for the synthesis of fatty acids, phospholipids, lipid A and other primary and secondary metabolites. ACP and ACP-like proteins are found throughout nature; however, differences between prokaryotic and eukaryotic ACP makes bacterial ACP-dependent enzymes attractive antibacterial targets. In the 50 years since its initial discovery, the structures of many bacterial ACPs (including that of Vibrio harveyi, the focus of this work) have been determined: all sharing a dynamic four α-helix bundle structure with a hydrophobic binding pocket enclosing attached fatty acyl chains. It has been hypothesized that ACP must undergo a conformational change to expose the sequestered acyl chain to a partner enzyme; the mechanism of and residues important to this conformational change remain unknown. Towards this end, I have explored the effect of constraining ACP’s termini on its conformational stability and function. Employing split-intein technology, a cyclized version of ACP was constructed. Using various biophysical techniques, I demonstrated that cyclic ACP was stabilized in the folded conformation in vitro relative to its linear counterpart. Furthermore, in vivo complementation assays proved that, counter to the prevailing hypothesis mentioned above, cyclic ACP can functionally replace the linear wild-type protein and support growth of an E. coli ACP-null strain. Additionally, I have expanded the use of fluorescence methods for studying ACP conformation, dynamics, and interaction with partner enzymes. Previous work in our lab established the use of Trp as a fluorescent probe of ACP conformation. To extend the utility of fluorescence to our lab’s extensive mutant ACP collection, the lone intrinsic tyrosine (Tyr 71) was tested for efficacy as a probe of ACP’s conformation. Although Tyr 71 was sensitive to its environment and to conformational change in ACP mutants, several experimental issues likely preclude its use as a fluorescent probe. To study ACP-partner enzyme interactions, two enzymes that lack endogenous Trp, E. coli UDP-N-acetylglucosamine acyltransferase (LpxA) and V. fischeri holo-ACP synthase (AcpS) were chosen for Trp-substitution at various positions. Characterization of these LpxAs suggests that Trp-substitution is a good probe for measuring ACP-enzyme interactions and transfer of the acyl chain to a partner enzyme’s active site.