Purification of N-methylputrescine oxidase from Nicotiana rustica

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

“…This might have been due to the instability of the enzyme under the high salt concentrations used in the procedure. This is consistent with the low yield and reported instability of MPO during orthodox chromatographic procedures (Haslam and Young 1992;Davies et al 1989) and is in contrast to diamine oxidase whose relative stability has led to its purification to homogeneity in as few as two chromatographic steps (Glatz et al 1987).…”

“…By comparison with molecular-weight standards we have calculated that MPO has an apparent sub-unit molecular weight of 53 kDa. This size is in reasonable agreement with that of the 54-kDa enzyme purified from N. rustica (Haslam and Young 1992) but smaller than the 70 kDa estimated from a partially purified extract of tobacco roots by an indirect method involving tagging MPO with a radiolabelled suicide inhibitor followed by detection in slices of an SDS-PAGE gel (Davies et al t989). It would also make it the smallest member of the diamine-oxidase family whose sub-unit molecular weights range from 61.5 to 112 kDa.…”

“…Purification of MPO to homogeniety has been reported from cultured roots of Nicotiana rustica L. (Haslam and Young 1992) and Hyoscyamus niger L. where it was Other esters Scopolamine 0~.~ fNCr{3 ~"nO-T r ooot e Fig. 1.…”

Molecular regulation of nicotine biosynthesis

“…The DAOs involved in nicotine and tropane alkaloid biosynthesis have higher affinity for Nmethylputrescine than for putresine and other symmetrical diamines (Haslam and Young 1992;Hashimoto et al 1990;Walton and McLauchlan 1990). In contrast, pea and pig DAOs bind N-methylputrescine with low affinity.…”

Alkaloids

“…Putrescine N-methyl transferase now has been cloned from a variety of plant species (141)(142)(143), and site-directed mutagenesis and homology models have led to insights into the structure function relationships of this enzyme (143). N-methylputrescine then is oxidized by a diamine oxidase to form 4-methylaminobutanal, which then spontaneously cyclizes to form the N-methyl-D-pyrrolinium ion (144,145). This enzyme, which has been cloned, seems to be a copper-dependent amine oxidase (146,147).…”