One-pot chemo-enzymatic synthesis of reporter-modified proteins

Worthington, Andrew S.; Burkart, Michael D.

doi:10.1039/b512735a

Cited by 125 publications

(173 citation statements)

References 14 publications

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“…Several previous studies have used the MichaelisMenten equation to obtain kinetic parameters for both EcPanK I and SaPanK II acting on a range of substrates [7,13,14,16,[32][33][34]. In the present study we took special care to obtain all measurements during the initial phase of the reaction (< 10% substrate consumed), and found that EcPanK I has a hyperbolic activity profile only for Pan, whereas it shows sigmoidal saturation curves for the pantothenamides (Fig.…”

Section: Quantifying the Variations In Pank Kinetics: Constructing A mentioning

confidence: 99%

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

et al. 2014

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N-substituted pantothenamides are analogues of pantothenic acid, the vitamin precursor of CoA, and constitute a class of well-studied bacterial growth inhibitors that show potential as new antibacterial agents. Previous studies have highlighted the importance of pantothenate kinase (PanK; EC 2.7.1.33) (the first enzyme of CoA biosynthesis) in mediating pantothenamide-induced growth inhibition by one of two proposed mechanisms: first, by acting on the pantothenamides as alternate substrates (allowing their conversion into CoA antimetabolites, with subsequent effects on CoA-and acyl carrier protein-dependent processes) or, second, by being directly inhibited by them (causing a reduction in CoA biosynthesis). In the present study we used structurally modified pantothenamides to probe whether PanKs interact with these compounds in the same manner. We show that the three distinct types of eubacterial PanKs that are known to exist (PanK I , PanK II and PanK III ) respond very differently and, consequently, are responsible for determining the pantothenamide mode of action in each case: although the promiscuous PanK I enzymes accept them as substrates, the highly selective PanK III s are resistant to their inhibitory effects. Most unexpectedly, Staphylococcus aureus PanK (the only known example of a bacterial PanK II ) experiences uncompetitive inhibition in a manner that is described for the first time. In addition, we show that pantetheine, a CoA degradation product that closely resembles the pantothenamides, causes the same effect. This suggests that, in S. aureus, pantothenamides may act by usurping a previously unknown role of pantetheine in the regulation of CoA biosynthesis, and validates its PanK as a target for the development of new antistaphylococcal agents.

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Section: Quantifying the Variations In Pank Kinetics: Constructing A mentioning

confidence: 99%

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

et al. 2014

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“…5,16 The importance of CoA analogues not only stems from their antibacterial activity, but also from their demonstrated use as chemical biology tools and as mechanistic probes of CoAdependent enzymes. These applications include both in vitro and in-cell labelling of carrier proteins in polyketide and nonribosomal peptide synthesis, [17][18][19] as well as aminoglycoside resistance inhibitors activated by the CoA biosynthetic pathway. 20 Although a wide range of pantothenamide derivatives have been reported over the years, most of these derivatives are modified at the amine moiety and less is known about the effects of modifications at other positions.…”

Section: Introductionmentioning

confidence: 99%

Exploring structural motifs necessary for substrate binding in the active site of Escherichia coli pantothenate kinase

Awuah¹,

Ma²,

Hoegl³

et al. 2014

Bioorganic & Medicinal Chemistry

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The coenzyme A (CoA) biosynthetic enzymes have been used to produce various CoA analogues, including mechanistic probes of CoA-dependent enzymes such as those involved in fatty acid biosynthesis. These enzymes are also important for the activation of the pantothenamide class of antibacterial agents, and of a recently reported family of antibiotic resistance inhibitors. Herein we report a study on the selectivity of pantothenate kinase, the first and rate limiting step of CoA biosynthesis. A robust synthetic route was developed to allow rapid access to a small library of pantothenate analogs diversified at the β-alanine moiety, the carboxylate or the geminal dimethyl group. All derivatives were tested as substrates of Escherichia coli pantothenate kinase (EcPanK). Four derivatives, all N-aromatic pantothenamides, proved to be equivalent to the benchmark Npentylpantothenamide (N5-pan) as substrates of EcPanK, while two others, also with N-aromatic groups, were some of the best substrates reported for this enzyme. This collection of data provides insight for the future design of PanK substrates in the production of useful CoA analogues. Graphical abstractThe promiscuity of E. coli PanK has limits! We report the synthesis of pantothenate analogs diversified at the β-alanine moiety, the carboxylate or the geminal dimethyl group. Interestingly, some of these are among the best substrates reported for E. coli PanK.

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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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One-pot chemo-enzymatic synthesis of reporter-modified proteins

Cited by 125 publications

References 14 publications

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

Variation in pantothenate kinase type determines the pantothenamide mode of action and impacts on coenzyme A salvage biosynthesis

Exploring structural motifs necessary for substrate binding in the active site of Escherichia coli pantothenate kinase

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

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