Abstract
Acyl carrier protein (ACP) interacts with many different enzymes during the synthesis of fatty acids, phospholipids, and other specialized products in bacteria. To examine the structural and functional roles of amino acids previously implicated in interactions between the ACP polypeptide and fatty acids attached to the phosphopantetheine prosthetic group, recombinant Vibrio harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion proteins. Circular dichroism revealed that, unlike Escherichia coli ACP, V. harveyi-derived ACPs are unfolded at neutral pH in the absence of divalent cations; all except F50A and I54A recovered native conformation upon addition of MgCl(2). Mutant I54A was not processed to the holo form by ACP synthase. Some mutations significantly decreased catalytic efficiency of ACP fatty acylation by V. harveyi acyl-ACP synthetase relative to recombinant ACP, e.g. F50A (4%), I54L (20%), and I54V (31%), whereas others (V12G, Y71A, and A59G) had less effect. By contrast, all myristoylated ACPs examined were effective substrates for the luminescence-specific V. harveyi myristoyl-ACP thioesterase. Conformationally sensitive gel electrophoresis at pH 9 indicated that fatty acid attachment stabilizes mutant ACPs in a chain length-dependent manner, although stabilization was decreased for mutants F50A and A59G. Our results indicate that (i) residues Ile-54 and Phe-50 are important in maintaining native ACP conformation, (ii) residue Ala-59 may be directly involved in stabilization of ACP structure by acyl chain binding, and (iii) acyl-ACP synthetase requires native ACP conformation and involves interaction with fatty acid binding pocket residues, whereas myristoyl-ACP thioesterase is insensitive to acyl donor structure.
Highlights
It is best known for its central role as a carrier of acyl intermediates during fatty acid synthesis, bacterial acyl carrier protein (ACP)1 has important functions as a donor of activated fatty acyl groups during biosynthesis of phospholipids [3], lipid A [4], lipoic acid [5], acylated homoserine lactones involved in quorum sensing [6, 7], and protein toxins, such as hemolysin [8]
To examine the structural and functional roles of amino acids previously implicated in interactions between the Acyl carrier protein (ACP) polypeptide and fatty acids attached to the phosphopantetheine prosthetic group, recombinant Vibrio harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion proteins
Exceptions include the elegant experiments of Tang et al [40], who used this approach to map the region of ACP that interacts with the glucosyltransferase involved in membrane-derived oligosaccharide synthesis
Summary
It is best known for its central role as a carrier of acyl intermediates during fatty acid synthesis (reviewed in Refs. 1 and 2), bacterial acyl carrier protein (ACP) has important functions as a donor of activated fatty acyl groups during biosynthesis of phospholipids [3], lipid A [4], lipoic acid [5], acylated homoserine lactones involved in quorum sensing [6, 7], and protein toxins, such as hemolysin [8]. Two-dimensional NMR has revealed that E. coli ACP has a defined but flexible tertiary structure dominated by three major parallel ␣-helices located at residues 3–14 (I), 37–51 (II), and 65–75 (III) [13] These helices enclose a hydrophobic core along their length, providing a binding pocket for fatty acids, which are covalently attached by a thioester bond to the 4-phosphopantetheine prosthetic group located at Ser-36 [14]. V. harveyi and E. coli ACP amino acid sequences are 86% identical, and only one of the differences involves a nonconservative change in a helical region: Val instead of Gly at position in V. harveyi ACP. Harveyi and E. coli ACP amino acid sequences are 86% identical, and only one of the differences involves a nonconservative change in a helical region: Val instead of Gly at position in V. harveyi ACP These ACPs share many biophysical properties [29].
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