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J. Biol. Chem., Vol. 282, Issue 7, 4494-4503, February 16, 2007

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Neutralization of Acidic Residues in Helix II Stabilizes the Folded Conformation of Acyl Carrier Protein and Variably Alters Its Function with Different Enzymes^*

From the Atlantic Research Centre, Departments of Pediatrics and Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada

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 Mg²⁺ or upon fatty acylation. Mg²⁺ binding to sites A or B independently stabilized native ACP conformation, whereas mutant SA/SB was folded in the absence of Mg²⁺, 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.

Received for publication, August 28, 2006 , and in revised form, December 5, 2006.

^* This work was supported by the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Atlantic Research Centre, Dalhousie University, Rm C-305 Clinical Research Centre, 5849 University Ave., Halifax, Nova Scotia B3H 4H7, Canada. Tel.: 902-494-7084; Fax: 902-494-1394; E-mail: david.byers{at}dal.ca.

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