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Table 2 Summary of high-throughput assays for methyltransferase

From: Biotechnological applications of S-adenosyl-methionine-dependent methyltransferases for natural products biosynthesis and diversification

Target MT Detection molecule Modality class Enzyme or reagent/reporter Measurement Detection limit Dynamic range Throughput Remarks Refs.
Salicylic acid carboxyl methyltransferase SAH → Homocysteine Coupled-enzyme reaction SAH nucleosidase and S-ribosylhomocysteine lyase/5,5′-dithiobis-2-nitrobenzoic acid (DTNB, Ellman's reagent) Absorbance 20 µm SAH   Multi-well plate format For purified MTs Hendricks et al. (2004)
Putrescine N-methyltransferase SAH → Homocysteine Coupled-enzyme reaction SAH nucleosidase and S-ribosylhomocysteine lyase/DTNB, Ellman’s reagent Absorbance 20 µm SAH 0–115 µm SAH (R2 = 0.9963) Multi-well plate format SAM degradation leads to TNB production
For purified MTs
Biastoff et al. (2006)
Protein arginine N-methyltransferase SAH → hypoxanthin Coupled-enzyme reaction SAH nucleosidase, adenine deaminase Absorbance 10 µm substrate 0–100 µm SAH Multi-well plate format Interference from protein which has absorbance at 280 nm
For purified MTs
Dorgan et al. (2006)
Histone methyltransferase and M.haeiii SAH → ammonia Coupled-enzyme reaction SAH nucleosidase, adenine deaminase and glutamate dehydrogenase/NADPH Absorbance 170 nm SAH 0–7000 µm peptide concentration Multi-well plate format Ammonia contamination from reaction needs to be avoided
For purified MTs
Duchin et al. (2015)
Catechol-O-methyltransferase SAH → homocysteine Coupled-enzyme reaction SAH hydrolase/fluorescein–cystamine–methyl red (FL-S–S-MR) Fluorescence  ≤ 1 µm homocysteine 0–50 µm homocysteine (R2 = 0.995) Multi-well plate format For purified MTs Wang et al. (2005)
Salicylic acid methyltransferase SAH → H2O2 Coupled-enzyme reaction SAH nucleosidase, xanthine oxidase, and horseradish peroxidase enzymes/Amplex
Red (10-acetyl-3,7-dihydroxyphenoxazine)
Fluorescence 36 nm for salicylic acid 0–10 µm salicylic acid (R2 = 0.94) Multi-well plate format For purified MTs Akhtar et al. (2018)
Protein arginine methyltransferases, histone-lysine N-methyltransferase and a sarcosine/dimethylglycine N-methyltransfease SAH → SIH Coupled-enzyme reaction Deaminase TM0936 Absorbance 2.35 µm substrate (absorbance) 2.35–46.96 µm SAM Multi-well plate format interference from protein which has absorbance at 280 nm
For purified MTs
Burgos et al. (2017)
Protein arginine methyltransferases, histone-lysine N-methyltransferase and a sarcosine/dimethylglycine N-methyltransfease SAH → SIH Coupled-enzyme reaction Deaminase TM0936/S-8-aza-adenosyl-l-methionine (8-aza-SAM) Fluorescence 25 µm substrate Three logs of linear dynamic range Multi-well plate format 8-aza-SAM is a good substrate for most MT
For purified MTs
Histone methyltransferase SAH RNA aptamer/riboswitch 3,5-difluoro-4-hydroxybenzylidene imidazolinone (DFHBI) Fluorescence 75 nm SAH tested Tens of nanomolar to tens of micromolar Flow cytometry Bind to ATP abd NAD + in vitro, although it has low fluorescence level
In vivo assay
Su et al. (2016)
Phenylethanolamine N-methyltransrease (PNMT)
Acetylserotonine O-methyltransferase [Asmt]
SAH → cysteine Coupled-microbial growth Adaptive laboratory evolution Delete serine acetyltransferase (cysE), overexpress cystathionine-β-synthase (cys4) and cystathionine-γ-lyase (cys3) Absorbance    10 million cells per passage In vivo assay
False positive rate may be high, due to improvement in native MTs activity
Luo et al. (2019)
Catechol-O-methyltransferase Vanillate Transcription factor Caulobacter crescentus VanR-VanO Fluorescence 0.01 mm Vanillate 0.01–1 mm vanillate Flow cytometry In vivo assay
Only specific to vanillate synthesis
Kunjapur and Prather (2019)
Commercial kit SAH RNA aptamer/riboswitch Tb-Streptavidin and dylight650 Time-resolved fluorescence resonance energy transfer 0.6 nm SAH 0.6–2500 nm SAH Multi-well plate format For purified MTs  
Commercial kit SAH → ATP Coupled-enzyme reaction MTase-Glo reagent and MTase-Glo detection reagent Luminescence 50 nm SAH 0–10 µm SAH (R2 > 0.99) Multi-well plate format For purified MTs  
Commercial kit SAH → H2O2 Coupled-enzyme reaction SAH nucleosidase, adenine deaminase, xanthine oxidase, and horseradish peroxidase enzymes/amplex
Red (10-acetyl-3,7-dihydroxyphenoxazine)
Fluorescence 1.25 µm Resorufin 0–10 µm Resorufin Multi-well plate format For purified MTs  
Commercial kit SAH Antibody SAH-d2 and Lumi4-tb cryptate conjugated antibody Time-resolved fluorescence resonance energy transfer 30 nm SAH 10–1000 nm SAH Multi-well plate format For purified MTs Kimos et al. (2016)