Gating mechanism of elongating β-ketoacyl-ACP synthases

Abstract

Carbon-carbon bond forming reactions are essential transformations in natural product biosynthesis. During de novo fatty acid and polyketide biosynthesis, β-ketoacyl-acyl carrier protein (ACP) synthases (KS), catalyze this process via a decarboxylative Claisen-like condensation reaction. KSs must recognize multiple chemically distinct ACPs and choreograph a ping-pong mechanism, often in an iterative fashion. Here, we report crystal structures of substrate mimetic bearing ACPs in complex with the elongating KSs from Escherichia coli, FabF and FabB, in order to better understand the stereochemical features governing substrate discrimination by KSs. Complemented by molecular dynamics (MD) simulations and mutagenesis studies, these structures reveal conformational states accessed during KS catalysis. These data taken together support a gating mechanism that regulates acyl-ACP binding and substrate delivery to the KS active site. Two active site loops undergo large conformational excursions during this dynamic gating mechanism and are likely evolutionarily conserved features in elongating KSs.

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases

“…The carbonyl oxygen of the substrate mimetic coordinates to His303 and His340, with the latter histidine serving an essential role for the condensation reaction. 32,33 Similar probe-KS interactions were observed in the crystal structures of FabB-AcpP 23,24 and IgaKS-IgaACP 59 , indicating an evolutionarily conserved PPant binding site. The acylated C12-FabF structure (PDB: 2GFY) overlays with the KS domain from C8Cl-AcpP=FabF with an RMSD of 0.314 Å ( Figure 4F).…”

“…AcpP loaded with the trans-C8-chloroacrylate PPant probe, C8Cl-crypto-AcpP (C8-AcpP) ( Figure 3), requires several hours to crosslink to FabF, but when loaded with an α-bromo crosslinker, such as the C16:1-α-bromo probe in this study, AcpP crosslinks in minutes. 24 This significant difference in crosslinking rates and the propensity for α-bromo crosslinkers to trap FabF in the gate-open conformation led us to hypothesize that crosslinking to trans-C8-chloroacrylate may require a slow, stochastic conformational change to the closed form to react with the active site (which was not certified by peer review) is the author/funder. All rights reserved.…”

“…19,20 ACPs must form transient, productive protein-protein interactions (PPIs) with each respective partner enzyme (PE) in order to deliver their substrates in catalytic competent forms. [21][22][23][24][25][26][27][28][29][30] KS-mediated carbon-carbon bond formation is the primary driving force of FAB. These enzymes use a two-step kinetic ping-pong bi-bi mechanism that can be viewed mechanistically as transacylation and condensation reactions ( Figure 1A).…”

“…32,[34][35][36] In our previous work, we determined crystal structures of FabB and FabF crosslinked to E. coli ACP (AcpP) loaded with either C12-or C16-α-bromo-pantetheinamide substrate mimetics. 24 These structures, along with biochemical and computational studies, led to the proposal of a double drawbridge-like gating mechanism comprised of two active site loops, loop 1 and loop 2, that undergo conformational rearrangements to regulate KS substrate recognition and processing ( Figure 1B). During substrate delivery, the gate opens to allow access to the KS active site, and in doing so, disrupts the oxyanion hole ( Figure 1B).…”

Structure and Dynamic Basis of Molecular Recognition Between Acyltransferase and Carrier Protein inE. coliFatty Acid Synthesis

“…Here we describe the first type II FAS ACP-AT complex solved to date. Similar to all crosslinked AcpP-partner protein structures available,[46][47][48][49] electrostatic complementarity between AcpP and FabD facilitates molecular recognition, (Figure S6) with the negatively charged AcpP interacting with a positive patch on FabD…”