One-pot chemoenzymatic preparation of coenzyme A analogues

Nazi, Ishac; Koteva, Kalinka; Wright, Gerard D.

doi:10.1016/j.ab.2003.09.005

Cited by 52 publications

(74 citation statements)

References 19 publications

(21 reference statements)

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“…The native molecular mass of MM2281 was estimated by gel filtration chromatography as previously described [6]. E. coli PS [6] and E. coli PANK [28] were overexpressed and purified as previously described. Protein concentrations were determined using the Bradford protein assay kit (Bio-Rad, Munich, Germany) with BSA as standard.…”

Section: Methodsmentioning

confidence: 99%

A novel isoform of pantothenate synthetase in the Archaea

2008

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Pantothenate is the essential precursor to CoA, which is of central importance for all parts of metabolism. This is shown by the fact that more than 400 enzymecatalyzed reactions are known to involve CoA (KEGG database [1]). Many more enzymes utilize acylated forms of CoA or require the CoA-derived phosphopantetheine as a prosthetic group. Typically, plants, fungi and microorganisms are able to synthesize pantothenate de novo, whereas animals rely on pantothenate in their diet.Pantothenate synthetase (PS) catalyzes the last step in the biosynthesis of pantothenic acid, also known as vitamin B 5 . The enzyme (EC 6.3.2.1) has been extensively studied in Escherichia coli [2,3], Mycobacterium tuberculosis [4,5], and Arabidopsis thaliana [6], and is highly conserved in the Bacteria and Eukaryota. Bacterial PS (Eqn 1) generates pantothenate from pantoate and b-alanine. It is an AMP-forming synthetase that proceeds via an acyl-adenylate intermediate and belongs to the HIGH superfamily of nucleotidyltransferases [3].

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Section: Methodsmentioning

confidence: 99%

A novel isoform of pantothenate synthetase in the Archaea

2008

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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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“…The His-tagged proteins were then obtained by the protocol of Nazi et al [18] as developed for the E. coli enzyme. The activity of DPCK-Aa was 145.5 units per mg protein and the identities of the products were verified by HPLC demonstration of phosphorylation of dephosphorylated CoA compounds isolated from E. coli.…”

Section: Production Of Dpckmentioning

confidence: 99%

“…3) Since no commercial source of DPCK is available, we next turned to preparation of this reagent. The bifunctional human DPCK/phosphopantetheine adenylyltransferase (PPAT) was difficult to reproducibly purify in an active form and thus we turned to E. coli DPCK which is encoded by the coaE gene and has been purified and characterized by Mishra et al [12] and by Nazi et al, [18]. A version of the gene encoding an N-terminally His-tagged version of DPCK was expressed in the standard phage T7 RNA polymerase expression system and the enzyme was readily purified in large quantities.…”

Section: Production and Analysis Of 33 P-labeled Coa And Coa Thioestementioning

confidence: 99%

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

Wadler

Cronan

2007

Analytical Biochemistry

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A new approach to determine in vivo pools of coenzyme A and short chain acyl-CoA thioesters is reported. The metabolites released by extraction with trichloroacetic acid are recovered and quantitatively dephosphorylated by treatment with shrimp alkaline phosphatase. Following phosphatase removal, the dephosphorylated CoA metabolites are quantitatively rephosphorylated by treatment with γ-labeled 33 P-ATP plus a dephospho-CoA kinase. The resulting radioactive CoA metabolites are then separated by reverse-phase high-performance liquid chromatography and quantitated by scintillation counting. Due to the enzymatic radio-phosphorylation, the assay is specific for CoA and its short chain thioesters and sensitive to subpicomole levels of these compounds.

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One-pot chemoenzymatic preparation of coenzyme A analogues

Cited by 52 publications

References 19 publications

A novel isoform of pantothenate synthetase in the Archaea

A novel isoform of pantothenate synthetase in the Archaea

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

Dephospho-CoA kinase provides a rapid and sensitive radiochemical assay for coenzyme A and its thioesters

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