Many proteins work in complexes(1), but few are as gigantic and contain a whole synthesis pathway comprising about 40 steps like the 2.6 megadalton FAS complex in fungi. It consists of more than 23,000 amino-acids and is responsible for the vital synthesis of fatty acids. Scientists working in ETH Zurich Professor Nenad Ban’s group at the Institute for Molecular Biology and Biophysics (2), who had already published the first clarification of the architecture of the fatty acid synthase from fungi and mammals at a resolution of about 5 angstrom in two “Science” papers last year (3), now present far more detailed analyses at 3.1 angstrom for the complex from yeast (4). These papers provide information about the structure of the various active sites of the individual enzyme components, the substrate specificity, catalytic mechanisms and substrate transport in the complex. With their more accurate analysis of the entire architecture and elucidation of the mechanism by which the resulting fatty acids are transported between the production sites during the synthesis, the ETH Zurich scientists have again achieved the feat of publishing two papers in the same issue of the scientific journal “Science” (5).

Insight into the inner workings of the FAS barrel

Looking at the fatty acid synthase enzyme as a whole, its shape resembles a barrel or ellipsoid (Figure 1). This in turn consists of two domes whose interiors are separated from one another by a central disk. However, the barrel is not entirely leak-tight: the domes have several openings to the exterior and there are also six in the disk. These openings enable substrate to diffuse into the two reaction chambers inside the domes where the synthesis takes place.

The atomic structure of the fatty acid synthase enzyme, which is even more detailed than the barrel-like rough structure just mentioned, is based on measurements using the Swiss Light Source at the Paul Scherrer Institute(6). Simon Jenni, a doctoral student in Nenad Ban’s team, explains that “Because of the opportunity to measure our crystals of fatty acid synthase at one of the world’s best X-ray sources, we succeeded after five years of work in computing excellent quality electron density maps and in constructing an atomic model of the fatty acid synthase.” For example this enabled the researchers to describe and compare the ketoacyl synthase accurately. This enzyme is embedded in the central disk, and its catalytic clefts open in the direction of the two dome cavities where it catalyses the condensation of the substrate. In contrast to certain bacteria, which need three variants, fungi make do with one version of the ketoacyl synthase enzyme.

A substrate transporter that unfolds and snaps shut several times

Altogether the ETH Zurich scientists were able to characterise the whole complex in this way. As a special structure they separately resolved the Acyl Carrier Protein (ACP) which is also a part of the complex and is responsible for substrate transport within the synthesis factory. They immobilised the structure of this transport protein in contact with the ketoacyl synthase, a switchblade inside the reaction cycle. Marc Leibundgut, a post-doc student in Nenad Ban’s group, explains that “Each ACP has a twin anchorage in the reaction chamber via flexible links that channel the free movement so that interactions with the individual enzymes occur preferentially, resulting in optimal catalysis.” (Figure 2).

Based on the structure and the situation that has now been determined, the researchers were able to infer a specific synthesis mechanism: a fatty acid precursor with two carbon atoms is loaded onto the ACP. Next the carrier protein pivots around in the reaction dome, thus bringing its charge to the various catalytic sites. In this respect the researchers compare the ACP to a switchblade knife that unfolds at each reactive cleft and presents the growing fatty acid for the elongation and modification reactions. Several transport cycles finally lead to a fatty acid with 16 to 18 carbon atoms. Because the ACP binds its substrate by what is known as covalent bonding, i.e. strong bonding, the efficiency of the process is increased since there is no loss of intermediate by diffusion out of the reaction chamber.

Figure 1: The three-dimensional model of the fatty acid sythase of fungi magnified ten million times. The enzymatically active protein sections are marked in different colours. (AT = acetyl transferase, green; ER = enoyl reductase, yellow; DH = dehydratase, orange; MPT = malonyl/palmitoyl transferase, red; ACP = acyl carrier protein, violet; KS = ketoacyl synthase, light blue; KR – ketoacyl reductase, dark blue). (Photo: Marc Leibundgut and Simon Jenni) large

Figure 2: The carrier protein ACP (shown diagrammatically as a light blue sphere) is anchored twice (green spheres) inside the reaction chamber by flexible bonds (yellow). ACP transports the fatty acid between the various reaction sites (red), where it is extended and/or modified. (© Science) large

More details, more therapy opportunities

The detailed structure of the fatty acid synthase represents a possible basis for finding specific targets to attack fungal diseases. This is particularly true because the structure of mammalian FAS is different to that of fungi. In addition, because fungal FAS is comparable with that of the mycobacteria, which cause diseases like leprosy and tuberculosis, here again there are potential starting points for the development of antibiotics.