CoA donates the phosphopantetheine arm, measuring approx. 18 Å, that is attached to the hydroxy group of the conserved serine residue in a Asp-Ser-Leu motif of ACP. The sulfhydryl in the prosthetic group is used to bind acyl chains via a thioester bond.
Figure 2Overview of the bacterial FA synthesis mechanism
After initiation of the acyl chain synthesis (reaction 3), the synthetic cycle is repeated multiple times until saturated C16 or C18 acyl-ACP is diverted for utilization in membrane biosynthesis. For the description of the reactions please refer to the text.
Fatty acyl chain extension occurs via the condensation reaction, in which ACP transfers its acyl chain on to one of the condensing enzymes FabB or FabF (step 1). The holo-ACP from this step is lost (step 2) and a malonyl-ACP enters, carrying the acyl chain extender unit. The malonyl group first loses a carbon dioxide (blue) and the growing acyl chain (red) is subsequently added onto the remaining two carbon groups attached to ACP (step 3). The resulting β-oxoacyl-ACP represents the first intermediate of a new fatty acyl elongation cycle. ‘R’ represents a saturated acyl chain of length 3+2n carbons. FabH catalyses only the very first condensation reaction that forms β-oxobutyryl-ACP. In that case, the acetyl starter group from acetyl-CoA is bound by FabH and condensed with malonyl-ACP.
(A) Two UFA synthesis pathways are shown for E. coli (left) and S. pneumoniae (right). E. coli utilizes FabA, which has a dual dehydratase and isomerase activity, whereas S. pneumoniae has a dedicated cis/trans isomerase (FabM), both of which are specific for C10 acyl chains. (B) The structure of the resulting cis-3-decenoyl-ACP.
Figure 5Sample high-resolution type II acyl-ACP structures
E. coli butyryl- and decanoyl-ACPs (A and B respectively), as well as octadecanoyl-ACP from spinach (C), show the highly conserved ACP structural motif consisting of a four α-helix bundle. The spinach ACP NMR structures are an overlay of the five lowest energy conformations and illustrate that the octadecanoyl chain is too large to fit into the hydrophobic pocket in its entirety. The acyl chains and prosthetic groups are coloured according to atom type. A three-dimensional interactive structure of (B) is available online at http://www.BiochemJ.org/bj/430/0001/bj4300001add.htm.
Anionic and cationic residues are marked on E. coli ACP in red and blue respectively in (A) and (B). (B) The recognition helix, helix II, is highlighted, which contains a number of conserved anionic residues as well as two hydrophobic residues (white). (C) Electrostatic surface representations of ACP, the left panel being orientated in the same way as (A) and the right panel rotated by 180 °, displaying the highly anionic face.
Human (A), yeast (B) and rat (C) type I ACPs. Overall, the secondary structure is conserved with type II proteins but other differences are present. ACP from yeast possesses a second domain (blue) and the entrance to the hydrophobic pocket appears to be closed off in type I ACPs (compare red circles in B and C with Figure 5B). The opening is closed off by loop I, which is orientated more towards helix III (thick arrow, C) and by Arg2150 (spheres, C). Ser36 is highlighted in stick representation.
The structure of the B. subtilis ACPS–ACP complex shows the functionally relevant homotrimer of ACPS (blue) that binds ACP (coloured) between the interface of two dimers. (B) An expanded snapshot showing the electrostatic interactions between ACP (coloured from N- to C-terminal in red to white) and ACPS (blue). Anionic residues are shown in red. The β-strands of ACPS are transparent for clarity. (C) Structure of the docked complex between FabG and ACP (purple), which binds between helices II and III and across the dimer interface of FabG. The β-oxoacyl substrate (starred arrow) would have to be extracted from the internal hydrophobic pocket of ACP and directed into the active site of FabG next to the NADPH cofactors (spherical representation). For simplicity, only the dimer of FabG is shown instead of the tetramer.
(A) Crystal structure of the mammalian type I FAS complex in surface representation, coloured according to the proteins' subunits (B). The yellow arrow denotes the location of ACP, for which there was no electron density in the crystal structure. The broken arrows indicate the directions of FAS movement as was identified by EM (as explained in the main text). (B) The mammalian FAS is composed of two α-chains which each contain a complete set of proteins for FA synthesis. DH, dehydratase; ER, enoyl reductase; KR, oxoacyl reductase; KS, oxoacyl synthase; MAT, malonyl-/acetyl-CoA transacylase; TE, thioesterase.
Figure 10Structures of the fungal 2.6 MDa type I crystal structure
(A) The heterododecameric arrangement consisting of six α- and β-chains (lower diagram), resembling an overall barrel shape. (B) X-ray structures of the yeast protein reveal ACP (surface representation) locked into position at the oxoacyl synthase domains. (C) EM experiments captured ACP at the active sites of a number of enzymes, revealing the dynamic manner of substrate shuttling during FA synthesis. (C) reproduced from Gipson, P., Mills, D.J., Wouts, R., Grininger, M., Vonck, J. and Kuhlbrandt, W. (2010) Direct structural insight into the substrate-shuttling mechanism of yeast fatty acid synthase by electron cryomucroscopy. Proc. Natl. Acad. Sci. U.S.A. 107, 9164–9169; . AT, acetyl transferase; DH, dehydratase; ER, enoyl reductase; KR, oxoacyl reductase; KS, oxoacyl synthase; PT, phosphopantetheine transferase; TE, thioesterase.
Figure 11Carrier proteins from NRPS and PKS systems
The PCP from the tyrocidine NRPS system undergoes changes depending on whether apo- or holo-PCP is present (A–C). The A form shown in (A) is only used by apo-PCP, whereas the A/H form (B) is shared by the apo- and holo-proteins. The H form (C) is exclusive to the holo state. The structures are coloured in a continuous spectrum from red (N-terminus) to blue (C-terminus). (D) The 3-oxobutyl-ACP intermediate from the S. coelicolor actinorhodin PKS, displaying the canonical ACP fold. The 3-oxobutyl group, which does not enter the ACP pocket, is shown in stick representation.