Site-directed mutagenesis of acyl carrier protein (ACP) reveals amino acidresidues involved in ACP structure and acyl-ACP synthetase activity

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 pr eviously implicated in interactions between the ACP polypeptide and fatty a cids attached to the phosphopantetheine prosthetic group, recombinant Vibri o harveyi ACP and mutant derivatives of conserved residues Phe-50, Ile-54, Ala-59, and Tyr-71 were prepared from glutathione S-transferase fusion prot eins. Circular dichroism revealed that, unlike Escherichia coli ACP, V. har veyi-derived ACPs are unfolded at neutral pH in the absence of divalent cat ions; all except F50A and 154A recovered native conformation upon addition of MgCl2. Mutant 154A was not processed to the holo form by ACP synthase. S ome mutations significantly decreased catalytic efficiency of ACP fatty acy lation 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 effect ive substrates for the luminescence-specific V. harveyi myristoyl-ACP thioe sterase. Conformationally sensitive gel electrophoresis at pH 9 indicated t hat fatty acid attachment stabilizes mutant ACPs in a chain length-dependen t manner, although stabilization was decreased for mutants F50A and A59G. O ur results indicate that (i) residues Ile-54 and Phe-50 are important in ma intaining native ACP conformation, (ii) residue Ala-59 may be directly invo lved in stabilization of ACP structure by acyl chain binding, and (iii) acy l-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.