Structure and conformational dynamics of fatty acid synthases

Multistep reactions rely on substrate channeling between active sites. Carrier protein-based enzyme systems constitute the most versatile class of shuttling systems due to their capability of linking multiple catalytic centers. In eukaryotes and some bacteria, these systems have evolved to multifunctional enzymes, which integrate all functional domains involved into one or more giant polypeptide chains. The metazoan fatty acid synthase (FAS) is a key paradigm for carrier protein-based multienzymes. It catalyzes the de novo biosynthesis of fatty acids from carbohydrate-derived precursors in more than 40 individual reactions steps. Its seven functional domains are encoded on one polypeptide chain, which assembles into an X-shaped dimer for activity. The dimer features two lateral reaction clefts, each equipped with a full set of active sites and a flexibly tethered carrier protein. Substrate loading and condensation in the condensing region are structurally and functionally separated from the β-carbon processing domains in the modifying region.
At the beginning of this thesis, only a single crystal structure of an intact metazoan FAS was known. FAS, in particular its modifying region, displays extensive conformational variability, according to electron microscopy (EM) studies. Thus, the aim was to obtain a crystal structure of the FAS modifying region to identify a ground-state structure of the FAS modifying region and to characterize its structural heterogeneity. The second aim was to establish a method for mapping conformational changes in multienzymes at high spatiotemporal resolution. Chapter 1 introduces FAS and gives a methodological overview of studying conformational dynamics of multienzymes.
In chapter 2, the 2.7-Å crystal structure of the entire 250-kDa modifying region of insect FAS is presented. It presents a conserved ground-state conformation adopted by the most divergent member of metazoan FAS. Remarkably, even the V-shape of the central dehydratase dimer is conserved, despite a minimal interface. Structural comparison to polyketide synthases (PKSs) highlights distinct properties of FAS such as strong domain interactions and the absence of an N-terminal β-α-β-α extension of the lateral non-catalytic pseudo-ketoreductase.
Chapter 3 presents a novel approach for identifying conformational dynamics of multienzymes in solution by filming with high-speed atomic force microscopy (HS-AFM) at a spatial resolution of 2-5 nm. The temporal resolution of 10 fps correlates with the timescale of large-scale conformational changes in FAS. Varied viewing angles are provided by combining different molecular tethering strategies. Reference-free particle classification enables the quantitative characterization of conformational states and their transitions.
Chapter 4 discusses the implications of the results on multienzyme biology with respect to biological questions and biotechnological applications. The results of this thesis provide the tools to understand the role of conformational dynamics in multienzymes with respect to their function. These findings will ultimately advance the engineering of enzymatic assembly lines for tailored compound production.