ISSN:
1432-2048
Keywords:
Datura
;
Cadaverine
;
Putrescine-N-methyltransferase
;
Root culture (Agrobacterium-transformed)
;
Tropane alkaloid
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract Putrescine-N-methyltransferase (PMT; EC 2.1.1.53), the first enzyme in the biosynthetic pathway leading from putrescine to tropane and pyrrolidine alkaloids, has been purified about 700-fold from root cultures of Datura stramonium established following genetic transformation with Agrabacterium rhizogenes. The native enzyme had a molecular weight estimated by gel-permeation chromatography on Superose-6 of 40 kDa; sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the peak fractions from Superose-6 chromatography revealed a band of 36 kDa molecular weight. Kinetic studies of the purified enzyme gave K m values for putrescine and S-adenosyl-l-methionine of 0.31 mM and 0.10 mM, respectively, and K i values for S-adenosyl-l-homocysteine and N-methylputrescine of 0.01 mM and 0.15 mM, respectively. The enzyme was active with some derivatives and analogous of putrescine, including 1,4-diamino-2-hydroxybutane and 1,4-diamino-trans-but-2-ene. Little activity was observed with 1,4-diamino-cis-but-2-ene and none with 1,3-diaminopropane or 1,5-diaminopentane (cadaverine), indicating a requirement for substrate activity of two amino groups in a trans conformation, separated by four carbon atoms. A large number of monoamines were inhibitors of the enzyme. Though not a substrate, cadaverine was a competitive inhibitor of the enzyme, with a K i of 0.04 mM; the significance of this in relation to the biosynthesis of cadaverine-derived alkaloids is discussed.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00191600
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