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

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

References

• 1 Dräger B. Chemistry and biology of calystegines.  Nat Prod Rep. 2004;  21 211-23
• 2 Mizusaki S, Tanabe Y, Noguchi M, Tamaki E. Phytochemical studies on tobacco alkaloids XIV. The occurrence and properties of putrescine N-methyltransferase in tobacco roots.  Plant Cell Physiol. 1971;  12 633-40
• 3 Flores H E, Galston A W. Analysis of polyamines in higher plants by high performance liquid chromatography.  Plant Physiol. 1982;  69 701-6
• 4 Stenzel O, Teuber M, Dräger B. Putrescine N-methyltransferase in Solanum tuberosum L., a calystegine-forming plant.  Planta. 2006;  223 200-12
• 5 Hendricks C L, Ross J R, Pichersky E, Noel J P, Zhou Z S. An enzyme-coupled colorimetric assay for S-adenosylmethionine-dependent methyltransferases.  Anal Biochem. 2004;  326 100-5
• 6 Hibi N, Fujita T, Hatano M, Hashimoto T, Yamada Y. Putrescine N-methyltransferase in cultured roots of Hyoscyamus albus. n-Butylamine as a potent inhibitor of the transferase both in vitro and in vivo .  Plant Physiol. 1992;  100 826-35
• 7 Zhu J, Dizin E, Hu X, Wavreille A -S, Park J, Pei D. S-Ribosylhomocysteinase (LuxS) is a mononuclear iron protein.  Biochemistry. 2003;  42 4717-26
• 8 Bradford M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding.  Anal Biochem. 1976;  72 248-54
• 9 Sambrock J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd edition Cold Spring Harbor; Cold Spring Harbor Laboratory Press 1989
• 10 Riddles P W, Blakeley R L, Zerner B. Reassessment of Ellman’s reagent.  Methods Enzymol. 1983;  91 49-60
• 11 Walton N J, Peerless A CJ, Robins R J, Rhodes M JC, Boswell H D, Robins D J. Purification and properties of putrescine N-methyltransferase from transformed roots of Datura stramonium L.  Planta. 1994;  193 9-15
• 12 Allart B, Gatel M, Guillerm D, Guillerm G. The catalytic mechanism of adenosylhomocysteine/methylthioadenosine nucleosidase from Escherichia coli. Chemical evidence for a transition state with a substantial oxocarbenium character.  Eur J Biochem. 1998;  256 155-62
• 13 Hoffman J L. Chromatographic analysis of the chiral and covalent instability of S-adenosyl-L-methionine.  Biochemistry. 1986;  25 4444-9
• 14 Feth F, Arfmann H -A, Wray V, Wagner K G. Determination of putrescine N-methyltransferase by high performance liquid chromatography.  Phytochemistry. 1985;  24 921-3
• 15 Moffatt B A, Weretilnyk E A. Sustaining S-adenosyl-L-methionine-dependent methyltransferase activity in plant cells.  Physiol Plant. 2001;  113 435-42
• 16 Heim W G, Jelesko J G. Association of diamine oxidase and S-adenosylhomocysteine hydrolase in Nicotiana tabacum extracts.  Plant Mol Biol. 2004;  56 299-308