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De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the Type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT—malonyl‐/acetyltransferase) and the condensation (KS—β‐ketoacyl synthase) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate‐binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.
Modularity is an aspect of a decomposable system with a coordinating authority that acts as a glue which holds the loosely held components. These multi-component entities (“modules”) facilitate rewiring into different designs allowing for change. Such modular character is a fundamental property of many biological entities, especially the family of megasynthases such as polyketide synthases (PKSs). The ability of these PKSs to produce diverse product spectra is strongly coupled to their broad architectural modularity. Decoding the molecular basis of modularity, i.e. identifying the folds and domains that comprise the modules as well as understanding constrains of the assembly of modules, is of utmost importance for harnessing megasynthases for the synthesis of designer compounds. In this study, we exploit the close semblance between PKSs and animal FAS to re-engineer animal FAS to probe the modularity of the FAS/PKS family. Guided by structural and sequence information, we truncate and dissect animal FAS into its components, and reassemble them to generate new PKS-like modules as well as bimodular constructs. The novel engineered modules resemble all four common module types of PKSs and demonstrate that this approach can be a powerful tool to create higher catalytic efficiency. Our data exemplify the inherent plasticity and robustness of the overall FAS/PKS fold, and open new avenues to explore FAS-based biosynthetic pathways for custom compound design.
De novo fatty acid biosynthesis in humans is accomplished by a multidomain protein, the type I fatty acid synthase (FAS). Although ubiquitously expressed in all tissues, fatty acid synthesis is not essential in normal healthy cells due to sufficient supply with fatty acids by the diet. However, FAS is overexpressed in cancer cells and correlates with tumor malignancy, which makes FAS an attractive selective therapeutic target in tumorigenesis. Herein, we present a crystal structure of the condensing part of murine FAS, highly homologous to human FAS, with octanoyl moieties covalently bound to the transferase (MAT) and the condensation (KS) domain. The MAT domain binds the octanoyl moiety in a novel (unique) conformation, which reflects the pronounced conformational dynamics of the substrate binding site responsible for the MAT substrate promiscuity. In contrast, the KS binding pocket just subtly adapts to the octanoyl moiety upon substrate binding. Besides the rigid domain structure, we found a positive cooperative effect in the substrate binding of the KS domain by a comprehensive enzyme kinetic study. These structural and mechanistic findings contribute significantly to our understanding of the mode of action of FAS and may guide future rational inhibitor designs.