Gating Mechanism of β-Ketoacyl-ACP Synthases

Abstract

Formation of carbon-carbon bonds via b-ketoacyl-acyl carrier protein (ACP) synthases (KS), are key reactions during de novo fatty acid and polyketide biosynthesis. KSs recognize multiple ACPs and choreograph ping-pong mechanisms often in an iterative fashion. Therefore, KSs must limit non-productive protein-protein interactions (PPIs) to achieve high degrees of reaction fidelity. To better understand the stereochemical features governing substrate discrimination during ACP•KS PPIs, we determined x-ray crystal structures complemented by molecular dynamic simulations of E. coli AcpP in complex with the elongating KSs, FabF and FabB. Covalently trapped substrate analogs were used to interrogate critical catalytic events accompanying carbon-carbon bond formation revealing a previously unknown gating mechanism during the binding and delivery of acyl-AcpPs. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features generally in elongating KSs. 3 Elaboration of these natural products is facilitated by a family of small ~8 kDa proteins, acyl carrier proteins (ACPs), that shuttle fatty acid and polyketide substrates and intermediates as they are processed by catalytic domains or discrete enzymes. 6,7 ACPs are translated in inactive apo forms and are posttranslationally modified to active holo forms with a 4'-phosphopantetheine (PPant) arm attached at a conserved serine residue, providing a thiol moiety that ligates substrates and intermediates to the ACPs. 6 Protein-protein interactions (PPIs) between ACPs and their enzymatic partners (or domains) regulate the catalytic activities of FASs and PKSs kinetically and stereochemically. 3,[8][9][10][11][12][13] Therefore, it is important to elucidate the structural and biophysical underpinnings of the PPIs between ACPs and their cognate KSs to understand reaction trajectories and product fidelity in FAS and PKS biosynthetic pathways.In contrast to initiating KSs 14,15 , which prime holo-ACPs, elongating KSs, can iteratively process fatty acid or polyketide intermediates. 3,16 Despite their distinct biosynthetic roles, these KSs operate via a common reaction mechanism, which can be separated into two halfreactions. 17,18 In the first half-reaction, an acyl-ACP associates with a KS, and its acyl cargo is transferred from the prosthetic PPant arm to a conserved, active site cysteine, producing a covalent acyl-KS intermediate and holo-ACP. Malonyl-ACP then replaces holo-ACP and undergoes a decarboxylative Claisen-like condensation with the acyl-KS adduct, producing and offloading β-ketoacyl-ACP 17 ( Figure S1). During the course of this ping-pong process, two distinct ACPs must sequentially interact with the KS enforcing exquisite temporal and spatial discrimination among subtly different chemical states of these acyl-ACPs.The Escherichia coli type II FAS system has served as a model for understanding ACPmediated PPIs as well as the enzymatic transformations catalyzed by FASs and PKSs ( Figure ...