Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent MethyltransferasesCitation formats

  • External authors:
  • M Kalim Akhtar
  • Dhanya Vijay
  • Saima Umbreen
  • Chris J McLean
  • Dominic J Campopiano
  • Gary J Loake

Standard

Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases. / Akhtar, M Kalim; Vijay, Dhanya; Umbreen, Saima; McLean, Chris J; Cai, Yizhi; Campopiano, Dominic J; Loake, Gary J.

In: Frontiers in Bioengineering and Biotechnology, Vol. 6, 2018, p. 146.

Research output: Contribution to journalArticlepeer-review

Harvard

Akhtar, MK, Vijay, D, Umbreen, S, McLean, CJ, Cai, Y, Campopiano, DJ & Loake, GJ 2018, 'Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases', Frontiers in Bioengineering and Biotechnology, vol. 6, pp. 146. https://doi.org/10.3389/fbioe.2018.00146

APA

Akhtar, M. K., Vijay, D., Umbreen, S., McLean, C. J., Cai, Y., Campopiano, D. J., & Loake, G. J. (2018). Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases. Frontiers in Bioengineering and Biotechnology, 6, 146. https://doi.org/10.3389/fbioe.2018.00146

Vancouver

Akhtar MK, Vijay D, Umbreen S, McLean CJ, Cai Y, Campopiano DJ et al. Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases. Frontiers in Bioengineering and Biotechnology. 2018;6:146. https://doi.org/10.3389/fbioe.2018.00146

Author

Akhtar, M Kalim ; Vijay, Dhanya ; Umbreen, Saima ; McLean, Chris J ; Cai, Yizhi ; Campopiano, Dominic J ; Loake, Gary J. / Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases. In: Frontiers in Bioengineering and Biotechnology. 2018 ; Vol. 6. pp. 146.

Bibtex

@article{e8e6c48c9b734883a6f5b798a198ea63,
title = "Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases",
abstract = "Methylated chemicals are widely used as key intermediates for the syntheses of pharmaceuticals, fragrances, flavors, biofuels and plastics. In nature, the process of methylation is commonly undertaken by a super-family of S-adenosyl methionine-dependent enzymes known as methyltransferases. Herein, we describe a novel high throughput enzyme-coupled assay for determining methyltransferase activites. Adenosylhomocysteine nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes were shown to function in tandem to generate a fluorescence signal in the presence of S-adenosyl-L-homocysteine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Since S-adenosyl-L-homocysteine is a key by-product of reactions catalyzed by S-adenosyl methionine-dependent methyltransferases, the coupling enzymes were used to assess the activities of EcoRI methyltransferase and a salicylic acid methyltransferase from Clarkia breweri in the presence of S-adenosyl methionine. For the EcoRI methyltransferase, the assay was sensitive enough to allow the monitoring of DNA methylation in the nanomolar range. In the case of the salicylic acid methyltransferase, detectable activity was observed for several substrates including salicylic acid, benzoic acid, 3-hydroxybenzoic acid, and vanillic acid. Additionally, the de novo synthesis of the relatively expensive and unstable cosubstrate, S-adenosyl methionine, catalyzed by methionine adenosyltransferase could be incorporated within the assay. Overall, the assay offers an excellent level of sensitivity that permits continuous and reliable monitoring of methyltransferase activities. We anticipate this assay will serve as a useful bioanalytical tool for the rapid screening of S-adenosyl methionine-dependent methyltransferase activities.",
author = "Akhtar, {M Kalim} and Dhanya Vijay and Saima Umbreen and McLean, {Chris J} and Yizhi Cai and Campopiano, {Dominic J} and Loake, {Gary J}",
year = "2018",
doi = "10.3389/fbioe.2018.00146",
language = "English",
volume = "6",
pages = "146",
journal = "Frontiers in Bioengineering and Biotechnology",
issn = "2296-4185",
publisher = "Frontiers Media S. A.",

}

RIS

TY - JOUR

T1 - Hydrogen Peroxide-Based Fluorometric Assay for Real-Time Monitoring of SAM-Dependent Methyltransferases

AU - Akhtar, M Kalim

AU - Vijay, Dhanya

AU - Umbreen, Saima

AU - McLean, Chris J

AU - Cai, Yizhi

AU - Campopiano, Dominic J

AU - Loake, Gary J

PY - 2018

Y1 - 2018

N2 - Methylated chemicals are widely used as key intermediates for the syntheses of pharmaceuticals, fragrances, flavors, biofuels and plastics. In nature, the process of methylation is commonly undertaken by a super-family of S-adenosyl methionine-dependent enzymes known as methyltransferases. Herein, we describe a novel high throughput enzyme-coupled assay for determining methyltransferase activites. Adenosylhomocysteine nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes were shown to function in tandem to generate a fluorescence signal in the presence of S-adenosyl-L-homocysteine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Since S-adenosyl-L-homocysteine is a key by-product of reactions catalyzed by S-adenosyl methionine-dependent methyltransferases, the coupling enzymes were used to assess the activities of EcoRI methyltransferase and a salicylic acid methyltransferase from Clarkia breweri in the presence of S-adenosyl methionine. For the EcoRI methyltransferase, the assay was sensitive enough to allow the monitoring of DNA methylation in the nanomolar range. In the case of the salicylic acid methyltransferase, detectable activity was observed for several substrates including salicylic acid, benzoic acid, 3-hydroxybenzoic acid, and vanillic acid. Additionally, the de novo synthesis of the relatively expensive and unstable cosubstrate, S-adenosyl methionine, catalyzed by methionine adenosyltransferase could be incorporated within the assay. Overall, the assay offers an excellent level of sensitivity that permits continuous and reliable monitoring of methyltransferase activities. We anticipate this assay will serve as a useful bioanalytical tool for the rapid screening of S-adenosyl methionine-dependent methyltransferase activities.

AB - Methylated chemicals are widely used as key intermediates for the syntheses of pharmaceuticals, fragrances, flavors, biofuels and plastics. In nature, the process of methylation is commonly undertaken by a super-family of S-adenosyl methionine-dependent enzymes known as methyltransferases. Herein, we describe a novel high throughput enzyme-coupled assay for determining methyltransferase activites. Adenosylhomocysteine nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes were shown to function in tandem to generate a fluorescence signal in the presence of S-adenosyl-L-homocysteine and Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine). Since S-adenosyl-L-homocysteine is a key by-product of reactions catalyzed by S-adenosyl methionine-dependent methyltransferases, the coupling enzymes were used to assess the activities of EcoRI methyltransferase and a salicylic acid methyltransferase from Clarkia breweri in the presence of S-adenosyl methionine. For the EcoRI methyltransferase, the assay was sensitive enough to allow the monitoring of DNA methylation in the nanomolar range. In the case of the salicylic acid methyltransferase, detectable activity was observed for several substrates including salicylic acid, benzoic acid, 3-hydroxybenzoic acid, and vanillic acid. Additionally, the de novo synthesis of the relatively expensive and unstable cosubstrate, S-adenosyl methionine, catalyzed by methionine adenosyltransferase could be incorporated within the assay. Overall, the assay offers an excellent level of sensitivity that permits continuous and reliable monitoring of methyltransferase activities. We anticipate this assay will serve as a useful bioanalytical tool for the rapid screening of S-adenosyl methionine-dependent methyltransferase activities.

U2 - 10.3389/fbioe.2018.00146

DO - 10.3389/fbioe.2018.00146

M3 - Article

C2 - 30406092

VL - 6

SP - 146

JO - Frontiers in Bioengineering and Biotechnology

JF - Frontiers in Bioengineering and Biotechnology

SN - 2296-4185

ER -