Structural and dynamical rationale for fatty acid unsaturation in
            <i>Escherichia coli</i>

Dodge, Greg J.; Patel, Ashay; Jaremko, Kara L.; McCammon, J. Andrew; Smith, Janet L.; Burkart, Michael D.

doi:10.1073/pnas.1818686116

Cited by 50 publications

(66 citation statements)

References 40 publications

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“…In FabF, AcpP1 and AcpP2 bury surface areas of 671.2 Å 2 and 675.4 Å 2 , respectively, while in FabB, AcpP1 and AcpP2 bury surface areas of 597.5 Å 2 and 607.4 Å 2 , respectively. These small contact areas are consistent with the transient nature of ACP interactions 10,23 . In addition, the differences in contact areas of AcpP-FabF and AcpP-FabB interfaces are consistent with the differing positions that AcpPs adopt relative to their KS partners ( Figure S2a Conformational heterogeneity in FabF active site loops.…”

Section: Introductionsupporting

confidence: 77%

Gating Mechanism of β-Ketoacyl-ACP Synthases

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Patel²,

Kim³

et al. 2019

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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 ...

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Section: Introductionsupporting

confidence: 77%

Gating Mechanism of β-Ketoacyl-ACP Synthases

Mindrebo

Patel²,

Kim³

et al. 2019

Preprint

Self Cite

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“…The E. coli type II FAS has served as a model system for understanding ACP-mediated PPIs and FAB 19,[29][30][31][32][33][34][35][36] . Here, we use this well-characterized system to better understand ACP•KS PPIs and KS substrate discrimination by structurally characterizing E. coli elongating KSs, FabB (KASI family KS), and FabF (KASII highlighting the importance of protein-protein interactions required at each step for substrate processing.…”

mentioning

confidence: 99%

Gating mechanism of elongating β-ketoacyl-ACP synthases

et al. 2020

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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.

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“…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…”

mentioning

confidence: 84%

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

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Mindrebo

Davis

et al. 2020

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Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP-AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP-AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the first x-ray crystal structure of a type II ACP-AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP-AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.Significance StatementThe essential role of acyltransferases (ATs) in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways, namely the selection and loading of starter and extender units onto acyl carrier proteins (ACPs), relies on catalytically productive ACP-AT interactions. Here, we describe and interrogate the first structure of a type II FAS malonyl-CoA:ACP-transacylase (MAT) in covalent complex with its cognate ACP. We combine structural, mutational, crosslinking and kinetic data with molecular dynamics simulations to describe a highly flexible and robust protein-protein interface, substrate-induced active site reorganization, and key subdomain motions that likely govern FAS function. These findings strengthen a mechanistic understanding of molecular recognitions between ACPs and partner enzymes and provide new insights for engineering AT-dependent biosynthetic pathways.

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Structural and dynamical rationale for fatty acid unsaturation in Escherichia coli

Cited by 50 publications

References 40 publications

Gating Mechanism of β-Ketoacyl-ACP Synthases

Gating Mechanism of β-Ketoacyl-ACP Synthases

Gating mechanism of elongating β-ketoacyl-ACP synthases

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

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