Sulfonyl 3-Alkynyl Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase

Ishikawa, Fumihiro; Haushalter, Robert W.; DJ, Lee; Finzel, Kara; Burkart, Michael D.

doi:10.1021/ja4042059

Cited by 38 publications

(47 citation statements)

References 39 publications

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“…[15] Chemical shift perturbations were observed in crypto-4-DMN-EcACP ( Fig. 3e-g, S9 and table S3) comparable to those previously observed for other sequestering crypto-EcACPs, [15] validating the location of the 4-DMN probe.…”

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confidence: 77%

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Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Beld¹,

Cang²,

Burkart³

2014

Angewandte Chemie

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In the fatty acid biosynthesis of plants and bacteria, the acyl carrier protein (ACP) is known to sequester elongating products within its hydrophobic core, but this dynamic mechanism remains poorly understood. In this paper we exploit solvatochromic pantetheine probes attached to ACP that fluoresce when sequestered. Addition of a catalytic partner lures the cargo out of the ACP and into the active site of the enzyme, enhancing fluorescence to reveal the elusive chain-flipping mechanism. This activity is confirmed by demonstration of a dual solvatochromic-crosslinking probe and solution-phase NMR. The chain-flipping mechanism can be visualized by single molecule fluorescent techniques, demonstrating specificity between the Escherichia coli ACP and its ketoacyl synthase catalytic partner KASII. Keywordssolvatochromism; acyl carrier protein; chain-flipping mechanism; fatty acid synthase; fatty acids Primary and secondary acetate metabolites are synthesized by dedicated fatty acid and polyketide synthases. Both families are subdivided in type I and type II architectures, based on whether they are encoded on one multidomain polypeptide chain or as separate proteins, respectively. These are often represented as molecular assembly lines with domains that perform chemistry on the growing metabolite while tethered to an acyl carrier protein (ACP), [1] which shuttles its cargo from one enzyme to the next. Although synthetic biologists have ambition to swap domains and modules between these synthases to create new molecular architectures, these efforts have been tempered by the discovery that both protein-substrate and protein-protein interactions play an important role in both iterative and

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confidence: 77%

“…Uniformly 15 N-labeled apo-EcACP was loaded with analog 1, forming cryptoEcACP. Cargo sequestration by acyl-EcACP elicits significant chemical shift perturbations in 15 N, 1 H-HSQC spectra compared to apo-and holo-EcACP. [15] Chemical shift perturbations were observed in crypto-4-DMN-EcACP ( Fig.…”

mentioning

confidence: 96%

Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Beld¹,

Cang²,

Burkart³

2014

Angewandte Chemie

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“…Significant effort towards engineering mechanism-based inhibitors has led to the development of the crosslinking probe used to study the ACP FabA interaction discussed previously in the ACP section (Figure 6c). 48, 158, 159 A sulfonyl 3-alkynyl pantetheinamide generates a reactive allene that forms a trapped covalent bond with the active site histidine of the dehydratase (Figure 6c). …”

Section: Fas Dehydration: Dehydratasesmentioning

confidence: 99%

Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering

2015

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Fatty acids are primary metabolites synthesized by complex, elegant, and essential biosynthetic machinery. Fatty acid synthases resemble an iterative assembly line, with an acyl carrier protein conveying the growing fatty acid to necessary enzymatic domains for modification. Each catalytic domain is a unique enzyme spanning a wide range of folds and structures. Although they harbor the same enzymatic activities, two different types of fatty acid synthase architectures are observed in nature. During recent years, strained petroleum supplies have driven interest in engineering organisms to either produce more fatty acids or specific high value products. Such efforts require a fundamental understanding of the enzymatic activities and regulation of fatty acid synthases. Despite more than one hundred years of research, we continue to learn new lessons about fatty acid synthases’ many intricate structural and regulatory elements. In this review, we summarize each enzymatic domain and discuss efforts to engineer fatty acid synthases, providing some clues to important challenges and opportunities in the field.

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“…Each of these materials can be optionally phosphorylated at the 4′-hydroxyl group of the pantetheine using a promiscuous pantetheine kinase (PanK) (Fig. 2) as given by the respective conversions 5a-5f into 6a-6f (12)(13)(14)(15). To obtain the phosphorylated mimetics 6a-6f, we incubated 5a-5f with PanK from the pantothenate (vitamin B 5 ) biosynthetic pathway in Escherichia coli (15).…”

Section: Resultsmentioning

confidence: 99%

Polyketide mimetics yield structural and mechanistic insights into product template domain function in nonreducing polyketide synthases

Barajas

Shakya

Moreno

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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Product template (PT) domains from fungal nonreducing polyketide synthases (NR-PKSs) are responsible for controlling the aldol cyclizations of poly-β-ketone intermediates assembled during the catalytic cycle. Our ability to understand the high regioselective control that PT domains exert is hindered by the inaccessibility of intrinsically unstable poly-β-ketones for in vitro studies. We describe here the crystallographic application of "atom replacement" mimetics in which isoxazole rings linked by thioethers mimic the alternating sites of carbonyls in the poly-β-ketone intermediates. We report the 1.8-Å cocrystal structure of the PksA PT domain from aflatoxin biosynthesis with a heptaketide mimetic tethered to a stably modified 4′-phosphopantetheine, which provides important empirical evidence for a previously proposed mechanism of PT-catalyzed cyclization. Key observations support the proposed deprotonation at C4 of the nascent polyketide by the catalytic His1345 and the role of a protein-coordinated water network to selectively activate the C9 carbonyl for nucleophilic addition. The importance of the 4′-phosphate at the distal end of the pantetheine arm is demonstrated to both facilitate delivery of the heptaketide mimetic deep into the PT active site and anchor one end of this linear array to precisely meter C4 into close proximity to the catalytic His1345. Additional structural features, docking simulations, and mutational experiments characterize proteinsubstrate mimic interactions, which likely play roles in orienting and stabilizing interactions during the native multistep catalytic cycle. These findings afford a view of a polyketide "atom-replaced" mimetic in a NR-PKS active site that could prove general for other PKS domains.polyketide synthase | polyketide mimetics | product template | aflatoxin | atom replacement P roduct template (PT) domains are a structural feature of the nonreducing class of iterative polyketide synthases (NR-PKSs). They both control the high reactivity of poly-β-ketone intermediates that are rapidly assembled upstream in the β-ketoacyl synthase (KS) domain (1) and catalyze their intramolecular closure to cyclic and fused cyclic products. How both of these tasks are carried out, and how competing self-condensations to more thermodynamically favored products are avoided, are central functional questions of NR-PKS catalysis. We address them here with the single PT domain for which an X-ray crystal structure exists and report the organized structure of a stable mimetic of the native but synthetically inaccessible poly-β-ketone substrate bound in the PksA PT domain.Biosynthesis of the environmental carcinogen aflatoxin B 1 is initiated by a fungal, iterative NR-PKS known as PksA (Fig. 1B) (2, 3). This polypeptide consists of six covalently linked enzyme domains, where the function of each has been characterized by its specialized role in the controlled polymerization of ketide units and cyclization to a particular product (Fig. 1A) (2, 3). Polyketide elongation is initiated by the sta...

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Sulfonyl 3-Alkynyl Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase

Cited by 38 publications

References 39 publications

Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Visualizing the Chain‐Flipping Mechanism in Fatty‐Acid Biosynthesis

Fatty acid biosynthesis revisited: structure elucidation and metabolic engineering

Polyketide mimetics yield structural and mechanistic insights into product template domain function in nonreducing polyketide synthases

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