Analysis and Engineering of Substrate Shuttling by the Acyl Carrier Protein (ACP) in Fatty Acid Synthases (FASs)

Abstract

ABSTRACTIn the large enzyme complexes of natural biosynthetic pathways, molecules are assembled like in a factory. Carrier domains shuttle substrates and intermediates as covalently attached cargo within the enzyme complex between active sites. The physical confinement of the reaction increases reaction rates and hinders pathway branching. Alternating interactions of substrate-loaded carrier domains with different catalytic domains modulate the chemical environment. In this study, we aim at assessing the impact of domain-domain interactions (DDIs) on the reaction progress of a multienzyme type I fatty acid synthase (FAS) in quantitative terms. We modulate DDIs by single interface mutations, and read out the impact on substrate shuttling by recording fatty acid (FA) chain length product spectra and FAS activities. Our data show that even single interface point mutations can severely affect FA synthesis. With molecular dynamics simulations and modeling, we relate the mutation effects to specific alterations in the molecular interaction networks and domain-domain binding energetics. Some of the presented mutations induce the synthesis of short-chain FAs. These compounds are important commodity products and potent precursors for microbial biofuel production.