Table 2.
Summary of high-throughput assays for methyltransferase
| 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) |