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A First Look at Yeast Fatty Acid Synthase Print

Fatty acids are the major constituents of eukaryotic and bacterial cellular membranes. They are used for functionally important post-translational protein modifications, and chains of fatty acids are the main storage compartments of an organism's chemical energy. Fatty acid synthesis is carried out by fatty acid sythase (FAS), which catalyzes cycles of multistep chemical reactions that are essentially the same in all organisms. FAS uses one acetyl-coenzyme A (CoA) and seven malonyl-CoA molecules to synthesize the 16-carbon palmitic acid, the most abundant fatty acid in eukaryotes. Now, for the first time, a group of researchers has determined the atomic structure of yeast Saccharomyces cerevisiae FAS derived from two crystals of the enzyme, using data collected at ALS Beamlines 8.2.1 and 8.2.2, as well as other synchrotron facilities.

Overall structure of yeast fatty acid synthase. (A) The barrel-shaped structure of FAS has two domes composed of β-subunit trimers (colored) and an equatorial wheel composed of α subunits (gray). (B) Two β-subunit trimers do not interact with each other (α subunits are omitted). (C) The top view of the β-subunit trimer showing it is formed by interactions between its N-terminal domains at the center and by interactions between neighboring subunits. (D) The α-subunit hexamer viewed from the top.

A Yeast Fatty Acid Factory

Saccharomyces cerevisiae is a well-studied species of budding yeast that has been known since ancient times, and used in baking bread and brewing beer. It is also one of our distant relatives. Like animals and plants, budding yeast are part of the eukaryotic domain. (It is estimated that humans share about 23% of our genome with this fungus.) S. cerevisiae is easy to culture and was one of the first eukaryotic genomes that was completely sequenced. It has been excellent model organism for studying the eukaryotic cell.

In recent years, S. cerevisiae has been used for studying eukaryotic fatty acid synthase (FAS), an enzymatic system that plays an essential role in embryogenesis and energy homeostasis. Now, two crystals of yeast FAS reveal this enzyme's extremely complex system: six reaction chambers of eight parts each (48 functional parts total), all working together to catalyze all reactions required for fatty acid synthesis in a particle with an atomic mass of 2.6 MDa (4.3–24 grams). Understanding how this fatty acid factory works will contribute to the development of antimicrobial, antifungal, antiobesity, and anticancer compounds.

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).