Acyl carrier protein (ACP), a small protein essential for bacterial growth and pathogenesis, interacts with diverse enzymes during the biosynthesis of fatty acids, phospholipids, and other specialized products such as lipid A. NMR and hydrodynamic studies have previously shown that divalent cations stabilize native helical ACP conformation by binding to conserved acidic residues at two sites (A and B) at either end of the "recognition" helix II. To examine the roles of these amino acids in ACP structure and function, site-directed mutagenesis was used to replace individual site A (Asp-30, Asp-35, Asp-38) and site B (Glu-47, Glu-53, Asp-56) residues in recombinant Vibrio harveyi ACP with the corresponding amides, along with combined mutations at each site (SA, SB) or both sites (SA/SB). Like native V. harveyi ACP, all individual mutants were unfolded at neutral pH but adopted a helical conformation in the presence of millimolar Mg 2؉ or upon fatty acylation. Mg 2؉ binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg 2؉ , suggesting that charge neutralization is largely responsible for ACP stabilization by divalent cations. Asp-35 in site A was critical for holo-ACP synthase activity, while acyl-ACP synthetase and UDP-N-acetylglucosamine acyltransferase (LpxA) activities were more affected by mutations in site B. Both sites were required for fatty acid synthase activity. Overall, our results indicate that divalent cation binding site mutations have predicted effects on ACP conformation but unpredicted and variable consequences on ACP function with different enzymes.
Acyl carrier protein (ACP)2 is an acidic and highly conserved protein typically consisting of 70 -100 residues and is essential for bacterial growth, communication, and pathogenesis. ACP is responsible for supplying acyl groups for the biosynthesis of a plethora of bacterial molecules, including fatty acids (1), phospholipids (2), lipid A (3), lipoic acid (4), hemolysin (5), acyl homoserine lactones involved in quorum-sensing (6), and the aldehyde substrate of luciferase in bioluminescent bacteria such as Vibrio harveyi (7). Other functions of ACP and its homologues include the production of membrane-derived oligosaccharides (8), rhizobial nodulation signaling factors (9), polyketide (10) and non-ribosomal peptide antibiotics (11), and lipoteichoic acid (12). The list of ACP-binding partners continues to expand based on proteomic efforts (13). The requirement for ACP in these diverse processes suggests that interactions between ACP and its partner enzymes must be specific. Information about how individual amino acid residues contribute to ACP conformation and interactions with functionally diverse enzymes will provide insight into the design of novel antimicrobial agents against ACP-dependent targets that are essential for bacterial growth and pathogenesis.Structural analyses of type II ACPs from Escherichia coli and several other bacterial species reveal a common three-helix bun...
