Colorimetric activity measurement of a recombinant putrescine N-methyltransferase from Datura stramonium

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 mu M for putrescine and 42 mu M for SAM, are lower than the previously reported values, due to alleviation of PMT inhibition by SAH.