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Planta Med 2006; 72(12): 1136-1141
DOI: 10.1055/s-2006-947191
Original Paper
Biochemistry and Molecular Biology
© Georg Thieme Verlag KG Stuttgart · New York

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

Stefan Biastoff1 , Michael Teuber1 , Zhaohui Sunny Zhou2 , Birgit Dräger1
  • 1Institute of Pharmaceutical Biology and Pharmacology, Martin-Luther-University Halle-Wittenberg, Halle/Saale, Germany
  • 2Department of Chemistry, Washington State University, Pullman, WA, USA
In memory of Professor Ernst Reinhard
Further Information

Publication History

Received: April 15, 2006

Accepted: June 1, 2006

Publication Date:
21 August 2006 (online)

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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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Prof. Dr. Birgit Dräger

Institute of Pharmaceutical Biology and Pharmacology

Martin-Luther-University Halle-Wittenberg

Hoher Weg 8

06120 Halle/Saale

Germany

Phone: +49-345-552-5765

Fax: +49-345-552-7021

Email: birgit.draeger@pharmazie.uni-halle.de