Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

Tallorin, Lorillee; Finzel, Kara; Nguyen, Quynh; Beld, Joris; Clair, James J. La; Burkart, Michael D.

doi:10.1021/jacs.5b13456

Cited by 22 publications

(20 citation statements)

References 34 publications

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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.…”

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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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confidence: 99%

Gating mechanism of elongating β-ketoacyl-ACP synthases

et al. 2020

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“…The acyl chain accesses the acyl binding pocket through a gate created by Phe201 and a flexible glycine-rich loop (residues 106-108) (Figure 3f, S5e), similar to the previously published dodecanoyl-FabB structure (PDB: 1EK4). 21 Only the C12AcpP-FabB structure reported herein illustrates a ACP-KS complex in a state preorganized to catalyze transacylation. The 2.50 Å C16AcpP-FabB structure reveals a lack of electron density for the C16-acyl chain in the acyl binding pocket, despite showing interpretable electron density for the 4'phosphopantetheineamide portion of the probe ( Figure S5f).…”

Section: Fabf Active Site Loops Adopt a "Gate-open" Conformation To Amentioning

confidence: 89%

Gating Mechanism of β-Ketoacyl-ACP Synthases

Mindrebo

Patel²,

Kim³

et al. 2019

Preprint

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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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“…The 6 tight binding of the ACP is quite remarkable because complexes of ACP with partner proteins have 7 typically been difficult to observe due to the transient and weak nature of their interactions (Cronan, 8 2014). Indeed, some of these complexes could only be obtained via crosslinking experiments 9 (Tallorin et al, 2016). Neither the ACP nor the four-helical bundle it associates with is present in 10 other GT-C glycosyltransferases, for which structural information is available.…”

Section: Role Of the Acyl Carrier Protein In Aftdmentioning

confidence: 99%

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

Tan

Zhang

Rodrigues

et al. 2019

Preprint

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SUMMARYMycobacterium tuberculosis causes tuberculosis, a disease that kills over one million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides – arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9 Å resolution structure of M. abscessus AftD determined by single particle cryo-electron microscopy. AftD has a conserved GT-C glycosyltransferase fold and three carbohydrate binding modules. Glycan array analysis shows that AftD binds complex arabinose glycans. Additionally, AftD is non-covalently complexed with an acyl carrier protein (ACP). 3.4 and 3.5 Å structures of a mutant with impaired ACP binding reveal a conformational change that suggests the ACP may regulate AftD function. Using a conditional knock-out constructed in M. smegmatis, mutagenesis experiments confirm the essentiality of the putative active site and the ACP binding for AftD function.

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Trapping of the Enoyl-Acyl Carrier Protein Reductase–Acyl Carrier Protein Interaction

Cited by 22 publications

References 34 publications

Gating mechanism of elongating β-ketoacyl-ACP synthases

Gating mechanism of elongating β-ketoacyl-ACP synthases

Gating Mechanism of β-Ketoacyl-ACP Synthases

Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria

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