Manipulating the Conformational Landscape of Large Macromolecular Complexes
by Benjamin Moritz Graf
Date of Examination:2022-04-01
Date of issue:2022-04-08
Advisor:Prof. Dr. Holger Stark
Referee:Prof. Dr. Holger Stark
Referee:Prof. Dr. Kai Tittmann
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EnglishThe de novo synthesis of fatty acids is one of the most central metabolic pathways in all forms of life. Among all known organisms, fatty acids are highly essential. Besides their role as energy resource, they are also required to maintain cellular growth, signal transduction processes and as main building block of biological membranes. Konrad Bloch and Feodor Lynen already described the metabolism of fatty acids in the early 1960s. Despite so many years of research surprisingly little is known about the enzymatic reactions involved in this process. In evolution two different fatty acid synthase (FAS) systems have evolved. Both generate fully saturated fatty-acyl chains. The type II system which can be found in plants, mitochondria, and most bacteria consists of different individual proteins. Other than that, the type I system of CMN-bacteria, higher metazoans, and fungi is a single large macromolecular protein complex that contains all required enzymatic domains. Within both systems, the growing fatty acyl chain is covalently bound to an acyl carrier protein (ACP) which mediates the transport between the different enzymatic domains. To ensure a complete processing of the covalent bound substrate, the ACP domain undergoes a highly dynamic process to interact with all distinct enzymatic domains. These ACP-enzyme interactions are transient which makes them infeasible for structure determination. However, detailed structural information of these ACP-enzyme interactions is necessary to elucidate the underlying biocatalytic mechanisms and their regulation on a molecular level. For the E. coli system other groups already successfully developed a biochemical tool that allows the specific trapping of substrate-bound ACP-enzyme interactions. Using X-ray crystallography, they managed to solve the structure of such normally transient interactions. Within this thesis, the strategy described for the E. coli system was successfully transferred to the type I FAS system of the baker’s yeast Saccharomyces cerevisiae. With a structure that shows the substrate-bound intermediate state of the transient ACP-keto synthase interaction we could proof, that this biochemical tool can also be applied to macromolecular eukaryotic FAS systems. Furthermore, the structure of this intermediate state provides an insight into the substrate binding and chain-length control mechanism within the keto synthase domain. A direct comparison with the bacterial homologue, the AcpP-FabB complex revealed several similarities between both systems. Additionally, the development of a chromatography-free purification protocol for the yeast FAS allowed us the discovery of a novel, yet unknown third subunit of the FAS complex. Structural and enzyme kinetic methods revealed that the newly discovered gamma-subunit plays a role in the kinetic regulation of the FAS. The binding of the gamma-subunit to the FAS molecule stabilizes a structural rearrangement of the overall complex from a non-rotated to a rotated state. In combination with steady-state enzyme kinetic measurements we showed that the gamma-subunit binding regulates the NADPH turnover by inducing a kinetic hysteresis effect and suppresses an uncontrolled futile cycle reaction at the FMN-bound enoyl reductase domain. Moreover, with the help of two amino acid sequences deriving from the y-subunit we managed to incorporate different single proteins into the FAS complex. This reveals the potential of the gamma-subunit as a biotechnological tool that could be used introduce additional fatty acyl specific activities into the FAS complex.
Keywords: Cryo-EM; X-ray crystallography; Fatty Acid Synthase; S. cerevisiae; Fatty Acid Synthesis; Crosslinking; Structural Dynamics; Macromolecular Complex