Evolution and mechanism of fatty acid synthase multienzymes

Fatty acids are central components of biological membranes, serve as energy storage compounds, and act as second messengers or as covalent modifiers governing protein localization. Biosynthesis of fatty acids uses a conserved mechanism across all species and is carried out in repeated cycles of reactions. In Eukaryotes, these reactions are catalyzed by type I fatty acid synthases (FAS), large architecturally diverse, multienzyme complexes that integrate all steps of fatty acid synthesis into complex biosynthetic assemblies. Two strikingly different types of FAS have emerged in fungi and in animals. The fungal FAS is a rigid, 2.6-MDa barrel- shaped structure with its 48 functional domains embedded in a matrix of scaffolding elements, which comprises almost 50% of the total sequence and determines the emergent multienzymes properties of fFAS. All functional core domains of fFAS are derived from monofunctional bacterial enzymes, but the evolutionary origin of the scaffolding elements remains enigmatic. In the first part of the thesis using a combined phylogenetic and structural biology approach we have identified two bacterial protein families of non-canonical fatty acid biosynthesis starter enzymes and trans-acting polyketide enoyl reductases (ER) as potential ancestors of core scaffolding regions in fFAS. The architectures of both protein families are revealed by representative crystal structures of the starter enzyme FabY and DfnA-ER. In both families, a striking structural conservation of insertions to scaffolding elements in fFAS is observed, despite marginal sequence identity. The combined phylogenetic and structural data provide first insights into the evolutionary origins of the complex multienzyme architecture of fFAS.
In contrast structural and evolutionarily analysis revealed that animal FAS is related to polyketide synthase type I (PKS I), which is utilized by bacteria to synthesize a broad spectrum of secondary metabolites. Animal FAS is
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an open X-shaped structure with catalytic domains not interrupted by the insertion of scaffolding elements but connected to each other via short not conserved linker sequences. Crystallographic data together with biochemical and electron microscopy (EM) analysis indicate that animal FAS displays an extraordinary degree of flexibility to ensure productive interactions between the active sites during the reaction cycle. Conformational changes most likely result from a combination of internal domain flexibility in the linker regions, which connects individual domains in the animal FAS. The second part of the thesis is thus dedicated to investigating how intra domain linking influences catalytic properties and conformational crosstalk between domains. This was achieved by generating more then 40 different constructs with various linker lenths. Combined structural and kinetic data from purified constructs helped us to better understand the emergent properties of the megasynthase system. A long-term goal is to use these insights for the construction of artificial multienzymes incorporating complete and complex molecular pathways.