Structure of Acyl Carrier Protein Bound to FabI, the FASII Enoyl Reductase from Escherichia coli

Abstract

Acyl carrier proteins play a central role in metabolism by transporting substrates in a wide variety of pathways including the biosynthesis of fatty acids and polyketides. However, despite their importance, there is a paucity of direct structural information concerning the interaction of ACPs with enzymes in these pathways. Here we report the structure of an acyl-ACP substrate bound to the Escherichia coli fatty acid biosynthesis enoyl reductase enzyme (FabI), based on a combination of x-ray crystallography and molecular dynamics simulation. The structural data are in agreement with kinetic studies on wild-type and mutant FabIs, and reveal that the complex is primarily stabilized by interactions between acidic residues in the ACP helix ␣2 and a patch of basic residues adjacent to the FabI substrate-binding loop. Unexpectedly, the acyl-pantetheine thioester carbonyl is not hydrogen-bonded to Tyr 156 , a conserved component of the short chain alcohol dehydrogenase/reductase superfamily active site triad. FabI is a proven target for drug discovery and the present structure provides insight into the molecular determinants that regulate the interaction of ACPs with target proteins. Acyl carrier proteins (ACPs)7 play an essential role in a diverse array of metabolic pathways including the biosynthesis of fatty acids (1, 2), polyketides (3), membrane-derived oligosaccharides (4), lipopolysaccharides (5, 6), and phospholipids (7). In each case the growing substrate is attached via a thioester to the ACP phosphopantetheine group. ACPs must therefore be able to recognize and interact, in an acyl group-dependent manner, with a wide variety of enzymes. In eukaryotic type I fatty acid synthesis (FASI) and in polyketide biosynthesis, the ACP occurs as part of a larger polypeptide that is also associated with other catalytic activities. In contrast, in bacterial type II fatty acid biosynthesis (FASII), each of the enzyme activities as well as the ACP are encoded by separate polypeptide chains (2). ACPs that function in FASII-mediated biosynthesis are small, highly soluble, acidic proteins that vary in molecular mass from 7.5 kDa (Escherichia coli) to 13 kDa (Mycobacterium tuberculosis) (1, 8 -11).Despite the central role that ACPs play in metabolism, structural details of their interaction with target proteins are sparse. Whereas the structures of ACPs from a variety of different species have been determined by x-ray crystallography (12) and NMR spectroscopy (see for example, Refs. 13 and 14), only one structure has been determined of ACP in complex with another protein, the holo-ACP synthase (AcpS) (15), and no structural information is available for the interaction between ACP and enzymes of the fatty acid biosynthesis pathway. AcpS attaches the phosphopantetheine to the ACP serine and thus, although valuable, the complex of AcpS and ACP differs fundamentally from other ACP-protein complexes and does not provide insight into the delivery of substrate by ACP.The NMR studies reveal that ACPs are highly flexible, a structural f...

Crystal structures and kinetic properties of enoyl‐acyl carrier protein reductase I from Candidatus Liberibacter asiaticus

“…9,14 In fact, in other ENR ternary complexes which include cofactor (NAD or NADP) and various inhibitors, the B-factors of cofactors and inhibitors are similar to their protein environment, indicating that the ternary structures are more stable than the binary structures. 14,16,32,33 Like other ENRs, ClFabI has the highly conserved signature sequence motif Tyr-(Xaa) 6 -Lys. In addition, another motif dyad Gly14-(Xaa) 5 -Ser20 and Val66 and Thr194 in ClFabI are also highly conserved and contained in the hydrogen bond network of FabIs 10,11 (Fig.…”

“…They both exist as dimer in solution, and all the disordered residues in apo-FabI crystals from other species are ordered in our apo-structure. Based on the sequence alignment and the residues involved in hydrogen bond network of FabIs, the signature sequence motif is expanded from Tyr-(Xaa) 6 -Lys to Gly-(Xaa) 5 -Ser-(Xaa) n -Val-Tyr-(Xaa) 6 -Lys-(Xaa) n -Thr. The resulting structure information regarding the active site pocket and details of the NAD 1 -binding mode could be used as bases for the rational design of more effective FabI inhibitors against Candidatus Liberibacter asiaticus.…”

“…FabI is crucial in the completion of cycles, particularly in the elongation phase of fatty acids synthesis. 6,7 Consequently, this important enzyme has recently attracted tremendous attention from researchers. Several crystal structures of FabI have been reported in other bacteria and protozoans, such as Escherichia coli, 8 Staphylococcus aureus, 9 Bacillus subtilis, 10 Bacillus cereus, 11 Helicobacter pylori, 12 Francisella tularensis, 13 Plasmodium falciparum, 14 and Mycobacterium tuberculosis.…”

“…16 Saito et al 17 elucidated the crystal structure of the enoyl-ACP reductase FabK (isoenzyme of FabI) in Streptococcus pneumonia and the binding mode of an inhibitor. Rafi et al 6 showed that complexes are primarily stabilized by interactions with acidic residues in the helix a2 of ACP, illustrating that FabI can serve as a target for drug discovery.…”

3-Substituted Indole Inhibitors Against Francisella tularensis FabI Identified by Structure-Based Virtual Screening

“…44 In E. coli FabI, the substrate binding loop has also been shown to become structured upon binding acyl carrier protein but in a slightly different conformation than that observed in the NAD + −triclosan complex. 41 Because the in silico screening began with a modeled loop between residues 192−205 the impact of this model on the inhibitor binding specificity was not known for the compounds selected from the in silico screening, thus kinetic methods were employed to examine inhibition of the NADH-bound form and the NAD + -bound form. The three inhibitors were found to be in rapid equilibrium and no evidence of slow-onset inhibition was apparent in progress curves (Supporting Information Figure S6).…”