Determining the yeast FAS structure was challenging because of its architectural complexity. The yeast FAS consists of 48 functional centers: six copies of eight independent functional domains in an α6β6 molecular complex of 2.6 MDa. Each of the α and β subunits contains four functional domains. These eight domains catalyze all reactions required for synthesis of fatty acids in yeast, which occurs in a limited space inside the α6β6 complex. The electron density maps had only a modestly high resolution (4 Å) but were of superb quality due to 9-fold noncrystallographic averaging in two crystal forms. Thus, visibility of side-chain electron density could be enhanced through sharpening, allowing the researchers to trace the entire polypeptide backbone for both chains and to position 50% of the protein side chains.

Reaction chambers of yeast fatty acid synthase. The FAS particle is shown in a surface representation and is sliced for a better view of the inside chambers with the ACP domain omitted. The positions of unique catalytic centers are represented by large balls, and the pathway traversed by ACP is presented as solid connecting lines. The arrows indicate the directions in the pathway. The small ball in the center (pink) represents the pivot about which ACP swings, and the broken lines indicate the orientations of the ACP in the pathway.

For the first time, the substrate-delivery and activating domains were visible. The substrate-delivery acyl carrier protein (ACP) resides within the FAS particle, while the activating phosphopantetheinyl transferase (PPT) domain lies outside. The six α subunits form a central wheel, and the β subunits form domes on the top and bottom of the wheel, creating six reaction chambers for fatty acid synthesis. Each of these functions independently as a fatty acid assembly line. The substrate-shuttling ACP traverses each chamber, behaving like a swinging arm and reaching six active sites. Surprisingly, the step at which the reactor is activated by attachment of the 4'-phosphopantetheine prosthetic group to the ACP must occur before complete assembly of the particle, since the PPT domain that attaches the arm lies outside the assembly, inaccessible to the ACP within.

The swinging motion of ACP is sufficient to deliver substrates to all of the active centers and results in a two-dimensional diffusion process for substrate delivery, significantly increasing the effective concentration of the substrates on top of that already achieved by the compartmentalization of the reactions in the FAS particle. There is now a complete framework for understanding the structural basis of this macromolecular machine’s important function.

Fatty acid assembly line. Six fatty acid assembly lines (reaction chambers) comprise the yeast fatty acid factory (FAS). The FAS particle is shown in a surface representation and is sliced for a better view of the inside chambers with the ACP domain omitted. Palmitoyl-CoA molecules (final product of yeast FAS), shown as balls, are leaving the structure through the pores around MPT domains.

In addition to elucidating the yeast FAS structure, the researchers were able to shed light on a three-decades-old hypothesis regarding the participation of a distant hydrophobic region in the recognition of palmitoyl residues. They found that a tunnel-like cavity, which lies near the active site of the malonyl transacylase (MPT) domain, might explain how the final product (palmitic/stearic acid) is transferred from ACP to CoA during the termination step. The composition of the tunnel reveals that only the palmitic/stearic acid chain is long enough to reach the hydrophobic region inside; consequently, it becomes a stable bound substrate of MPT.

Many applications can come out of an understanding of the FAS yeast structure. For example, the structure of the enoyl reductase (ER) domain of the FAS β subunit is the first one of the FabK-like ERs that is not sensitive to the antibiotic triclosan, an effective inhibitor of bacterial ER. Thus, the yeast FAS–ER structure can aid investigation into the source of this antibiotic resistance, enabling structure-based drug design to develop antifungal pharmaceuticals.

Research conducted by I.B. Lomakin and Y. Xiong (Yale University); and T.A. Steitz (Yale University and the Howard Hughes Medical Institute).

Research Funding: The Agouron Institute and the National Institutes of Health. Operation of the ALS is supported by the U.S. Department of Energy, Office of Basic Energy Sciences (BES).

Publication about this research: I.B. Lomakin, Y. Xiong, and T.A. Steitz, "The crystal structure of yeast fatty acid synthase, a cellular machine with eight active sites working together," Cell 129, 319 (2007).

ALSNews Vol. 282, November 28, 2007