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.
N1 - Funding Information:
We thank Dr. Andrew Collis and Dr. Nicola Crowhurst for their assistance throughout the project. This work was supported by an IBioIC Exemplar Project Award to GJL, DJC, and YC, and the UAEU Program for Advanced Research Fund (31S307) awarded to MKA.
Publisher Copyright:
© 2018 Akhtar, Vijay, Umbreen, McLean, Cai, Campopiano and Loake.
PY - 2018/10/18
Y1 - 2018/10/18
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.
KW - Amplex Red
KW - Biocatalysis
KW - Drug screening
KW - High throughput
KW - Methylation
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UR - http://www.scopus.com/inward/citedby.url?scp=85055730640&partnerID=8YFLogxK
U2 - 10.3389/fbioe.2018.00146
DO - 10.3389/fbioe.2018.00146
M3 - Article
AN - SCOPUS:85055730640
SN - 2296-4185
VL - 6
JO - Frontiers in Bioengineering and Biotechnology
JF - Frontiers in Bioengineering and Biotechnology
IS - OCT
M1 - 146
ER -