Key residues responsible for acyl carrier protein and beta-ketoacyl-acyl carrier protein reductase (FabG) interaction.
Fatty acid synthesis in bacteria is catalyzed by a set of individual enzymes collectively known as type II fatty-acid synthase. Each enzyme interacts with acyl carrier protein (ACP), which shuttles the pathway intermediates between the proteins. The type II enzymes do not possess primary sequence similarity that defines a common ACP-binding site, but rather are hypothesized to possess an electropositive/hydrophobic surface feature that interacts with the electronegative/hydrophobic residues along helix alpha2 of ACP (Zhang, Y.-M., Marrakchi, H., White, S. W., and Rock, C. O. (2003) J. Lipid Res. 44, 1-10). We tested this hypothesis by mutating two surface residues, Arg-129 and Arg-172, located in a hydrophobic patch adjacent to the active site entrance on beta-ketoacyl-ACP reductase (FabG). Enzymatic analysis showed that the mutant enzymes were compromised in their ability to utilize ACP thioester substrates but were fully active in assays with a substrate analog. Direct binding assays and competitive inhibition experiments showed that the FabG mutant proteins had reduced affinities for ACP. Chemical shift perturbation protein NMR experiments showed that FabG-ACP interactions occurred along the length of ACP helix alpha2 and extended into the adjacent loop-2 region to involve Ile-54. These data confirm a role for the highly conserved electronegative/hydrophobic residues along ACP helix alpha2 in recognizing a constellation of Arg residues embedded in a hydrophobic patch on the surface of its partner enzymes, and reveal a role for the loop-2 region in the conformational change associated with ACP binding. The specific FabG-ACP interactions involve the most conserved ACP residues, which accounts for the ability of ACPs and the type II proteins from different species to function interchangeably.
Zhang
Yong-Mei
YM
Department of Infectious Diseases, St. Jude Children's Research Hospital, Memphis, Tennessee 38105, USA.
Wu
Bainan
B
Zheng
Jie
J
Rock
Charles O
CO
D000213:Acyl Carrier Protein / Q000737:chemistry*; D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000502:physiology; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism*; D007532:Isoleucine / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D020349:Surface Plasmon Resonance; D013997:Time Factors
D000213:Acyl Carrier Protein; D007532:Isoleucine; D001120:Arginine; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase