Synthesis and application of isotopically labeled flavin nucleotides

Mishanina, Tatiana V.; Kohen, Amnon

doi:10.1002/jlcr.3313

Cited by 10 publications

(15 citation statements)

References 33 publications

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“…To investigate, we turned to FDTS reconstituted with isotopically-labeled FAD ( 11 ). FDTS reconstituted with FAD labeled at the adenine moiety yielded no labeled intermediate, but the reaction of [7a,8a- 3 H]-FAD-FDTS produced a tritiated trapped intermediate (Fig.…”

Section: What Is the Base-modified Intermediate?mentioning

confidence: 99%

An unprecedented mechanism of nucleotide methylation in organisms containing thyX

Mishanina

Karunaratne

et al. 2016

Science

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In several human pathogens, thyX-encoded flavin-dependent thymidylate synthase (FDTS) catalyzes the last step in the biosynthesis of thymidylate, one of the four DNA nucleotides. ThyX is absent in humans, rendering FDTS an attractive antibiotic target; however, the lack of mechanistic understanding prohibits mechanism-based drug design. Here we report trapping and characterization of two consecutive intermediates, which together with previous crystal structures indicate that the enzyme’s reduced flavin relays a methylene from the folate carrier to the nucleotide acceptor. Furthermore, these results corroborate an unprecedented activation of the nucleotide that involves no covalent modification but only electrostatic polarization by the enzyme’s active site. These findings indicate a mechanism that is very different from thymidylate biosynthesis in humans, underscoring the promise of FDTS as an antibiotic target.

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Section: What Is the Base-modified Intermediate?mentioning

confidence: 99%

An unprecedented mechanism of nucleotide methylation in organisms containing thyX

Mishanina

Karunaratne

et al. 2016

Science

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“…Tris(hydroxymethyl)aminomethane (Tris) and urea (carbamide) were obtained from Research Products International Corp. Radio-and stable-isotope-labeled FAD cofactors were enzymatically synthesized from riboflavin and ATP, as previously described. 55 The FDTS from Thermatoga maritima (TM0449, GenBank accession number NP228259) was expressed and purified following the published procedure. 55 The FDTS was reconstituted with isotopically labeled flavins as detailed elsewhere.…”

Section: Fdts Kineticsmentioning

confidence: 99%

“…55 The FDTS from Thermatoga maritima (TM0449, GenBank accession number NP228259) was expressed and purified following the published procedure. 55 The FDTS was reconstituted with isotopically labeled flavins as detailed elsewhere. 55 63 .…”

Section: Fdts Kineticsmentioning

confidence: 99%

“…55 The FDTS was reconstituted with isotopically labeled flavins as detailed elsewhere. 55 63 . NMR spectra were processed using NMRPipe 64 and analyzed using NMRView.…”

Section: Fdts Kineticsmentioning

confidence: 99%

“…The acid-quench data from ref 46 Preparation and activity tests of 5-deaza-5-carba-FAD-FDTS Holo-FDTS was first treated with a saturated solution of NaCl as previously described 50,55 , to remove most of the bound FAD. This enzyme was then denatured with…”

Section: Data Fitting For Fdts-catalyzed Reaction Kineticsmentioning

confidence: 99%

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Probing the methylene and hydride transfers in flavin- dependent thymidylate synthase

Karunaratne¹

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In humans de novo synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP), an essential building block of DNA, utilizes an enzymatic pathway requiring thymidylate synthase (TSase) and dihydrofolate reductase (DHFR). The enzyme flavin-dependent thymidylate synthase (FDTS) represents an alternative enzymatic pathway to synthesize dTMP, which is not present in human cells. A number of pathogenic bacteria, however, depend on this enzyme in lieu of or in conjunction with the analogous human pathway. Thus, inhibitors of this enzyme may serve as antibiotics. Here, we review the similarities and differences of FDTS vs. TSase including aspects of their structure and chemical mechanism. In addition, we review current progress in the search for inhibitors of flavin dependent thymidylate synthase as potential novel therapeutics.

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Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors**

et al. 2023

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Flavin adenine dinucleotide (FAD) is an essential redox cofactor in cellular metabolism. The organic synthesis of FAD typically involves coupling flavin mononucleotide (FMN) with adenosine monophosphate, however, existing synthesis routes present limitations such as multiple steps, low yields, and/or difficult‐to‐obtain starting materials. In this study, we report the synthesis of FAD nucleobase analogues with guanine/cytosine/uracil in place of adenine and deoxyadenosine in place of adenosine using chemical and enzymatic approaches with readily available starting materials, achieved in 1–3 steps with moderate yields (10–57 %). We find that the enzymatic route using Methanocaldococcus jannaschii FMN adenylyltransferase (MjFMNAT) is versatile and can produce these FAD analogues in high yields. Further, we demonstrate that Escherichia coli glutathione reductase is capable of binding and using these analogues as cofactors. Finally, we show that FAD nucleobase analogues can be synthesized inside a cell from cellular substrates FMN and nucleoside triphosphates by the heterologous expression of MjFMNAT. This lays the foundation for their use in studying the molecular role of FAD in cellular metabolism and as biorthogonal reagents in biotechnology and synthetic biology.

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Synthesis and application of isotopically labeled flavin nucleotides

Cited by 10 publications

References 33 publications

An unprecedented mechanism of nucleotide methylation in organisms containing thyX

An unprecedented mechanism of nucleotide methylation in organisms containing thyX

Probing the methylene and hydride transfers in flavin- dependent thymidylate synthase

Efficient Chemical and Enzymatic Syntheses of FAD Nucleobase Analogues and Their Analysis as Enzyme Cofactors**

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