| dc.description | Acyl carrier protein (ACP) is a small protein that supplies fatty acyl groups to a remarkable diversity of enzymes and pathways, and is thus essential for bacterial growth and pathogenesis. A central acidic ACP helix (helix II) has been implicated in interaction with ACP partner enzymes, but the roles of specific ACP residues in enzyme docking and conformational changes to release the acyl group to the enzyme active site are largely unknown. Using recombinant Vibrio barvyi ACP (rACP) as a template for site-directed mutagenesis, I have explored the contributions of residues in and around helix II to ACP conformation and function with a variety of enzyme systems. An acidic residue at position 41 in the middle of helix II was found to be critical for the activity of fatty acid synthase, but not acyl-ACP synthetase. Combined neutralization of acidic residue clusters at either end of helix II (sites A and B) did not prevent stabilization of ACP conformation by divalent cation binding at the unaltered site, but differentially affected ACP function. Holo-ACP synthase was sensitive to mutations in site A, while site B mutations preferentially affected acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities. Tryptophan mutagenesis to provide site-specific fluorescent probes of local conformation revealed that ACP folds upon interaction with LpxA, and that ACP folding induced by divalent cation binding and by fatty acylation produce distinct environments in the hydrophobic core. Overall, my results reinforce the concept of helix II as the "recognition helix" of ACP, but also provide detailed insights into the relationships between ACP structure and function that are relevant to the regulation of lipid metabolism and the design of novel antibacterial drugs. | en_US |
