Mechanism of Substrate Shuttling by the Acyl-Carrier Protein within the Fatty Acid Mega-Synthase

Abstract

Fatty acid mega-synthases (FAS) are large complexes that integrate into a common protein scaffold all the enzymes required for the elongation of aliphatic chains. In fungi, FAS features two independent dome-shaped structures, each 3-fold symmetric, that serve as reaction chambers. Inside each chamber, three acyl-carrier proteins (ACP) are found double-tethered to the FAS scaffold by unstructured linkers; these are believed to shuttle the substrate among catalytic sites by a mechanism that is yet unknown. We present a computer-simulation study of the mechanism of ACP substrate-shuttling within the FAS reaction chamber, and a systematic assessment of the influence of several structural and energetic factors thereon. Contrary to earlier proposals, the ACP dynamics appear not to be hindered by the length or elasticity of the native linkers, nor to be confined in well-defined trajectories. Instead, each ACP domain may reach all catalytic sites within the reaction chamber, in a manner that is essentially stochastic. Nevertheless, the mechanism of ACP shuttling is clearly modulated by volume-exclusion effects due to molecular crowding and by electrostatic steering toward the chamber walls. Indeed, the probability of ACP encounters with equivalent catalytic sites was found to be asymmetric. We show how this intriguing asymmetry is an entropic phenomenon that arises from the steric hindrance posed by the ACP linkers when extended across the chamber. Altogether, these features provide a physically realistic rationale for the emergence of substrate-shuttling compartmentalization and for the apparent functional advantage of the spatial distribution of the catalytic centers.

Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering

“…37 Extensive computer simulation work found that each ACP bounces stochastically within this chamber, but with asymmetric probability observed for multiple ACPs to simultaneously interact with equivalent catalytic sites. 38 The authors conclude this to be an entropic phenomenon and hesitate to suggest cooperativity between subunits. The yeast type I FAS (PDB: 3HMJ) 39 offers a similar barrel shape with a different domain arrangement, further studied by electron microscopy (EM).…”

Engineering fungal de novo fatty acid synthesis for short chain fatty acid production

“…The fungal FAS is a highly efficient FA producing machinery that runs synthesis at higher turnover numbers than other type I FAS systems (18.2, 2.0 and 3.4 cycles per second; values per set of active sites calculated from specific activities reported for S. cerevisiae 10, Corynebacterium ammoniagenes 11 and chicken FAS12, respectively). The synthetic advantage of the fungal FAS lies in its highly developed architecture, in which the catalytic domains are embedded in an extensive scaffolding matrix, and substrate shuttling by the acyl carrier protein (ACP) is subtly balanced between electrostatic steering and molecular crowding properties131415 (Fig. 1a).…”

Strategies in megasynthase engineering – fatty acid synthases (FAS) as model proteins

“…This structural organization likely preserves linkers from interfering in ACP:domain interactions, and, concomitantly, may support the loading of the covalent acyl moiety by steering the acyl tail into the binding channels. A computational study, on the basis of S. cerevisiae FAS data, refined the understanding of ACP-mediated substrate shuttling in S. cerevisiae FAS by confirming steering in the sense of promoting correct orientations, as well as suggesting electrostatic steering by charge complementarity of the surfaces of binding partners [33]. Recent studies have characterized ACP of FAS megasynthases as not sequestering the covalently bound acyl moiety, which is supportive of molecular steering effects underlying substrate shuttling [34–35].…”