The Kinetics of Carbon-Carbon-Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the beta-ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon-carbon-bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here we report enzyme kinetics of the KS-mediated carbon-carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS-mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. Intriguingly, the KS protomers within the dimer exhibit positive cooperativity, investigated by mutational studies and acyl-carrier analysis, which likely serves the regulation of biosynthesis. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.