Colorimetric Activity Measurement of a Recombinant Putrescine N-Methyltransferase from Datura stramonium

Abstract

Putrescine N-methyltransferase (PMT, EC 2.1.1.53) catalyses the S-adenosyl-L-methionine (SAM or AdoMet)-dependent methylation of putrescine to N-methylputrescine within the biosynthetic pathways of calystegines, nicotine, and tropane alkaloids in medicinal plants and produces S-adenosyl-L-homocysteine (SAH or AdoHcy). Determination of PMT activity was time-consuming and hardly reproducible in the past because it required tedious separation steps after chemical derivatisation or radioactive labelling of N-methylputrescine. A convenient and accurate enzyme-coupled colorimetric assay is based on the conversion of SAH to homocysteine by 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTAN/SAHN, EC 3.2.2.9) and S-ribosylhomocysteine lyase (LuxS, EC 4.4.1.21). Homocysteine is quantified by 5,5′-dithiobis-2-nitrobenzoic acid. Putrescine was shown not to interfere with MTAN or LuxS. The colorimetric assay was validated by HPLC analysis. K _m values determined by the assay, 108 μM for putrescine and 42 μM for SAM, are lower than the previously reported values, due to alleviation of PMT inhibition by SAH.

Abbreviations

DTNB:5,5′-dithiobis-2-nitrobenzoic acid

LuxS:S-ribosylhomocysteine lyase

MTAN:5′-methylthioadenosine nucleosidase

PMT:putrescine N-methyltransferase

SAH:S-adenosyl-L-homocysteine

SAM:S-adenosyl-L-methionine

TNB:2-nitro-5-thiobenzoic acid

Key words

Putrescine N-methyltransferase - S-adenosyl-L-methionine - homocysteine - colorimetric assay - Datura stramonium - Solanaceae

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