Microsome-mediated transformation of O-methylandrocymbine to demecolcine and colchicine.

Microsomal preparations from immature seeds of Colchicum autumnale L. catalyse the ring expansion reaction of O-methylandrocymbine to demecolcine in the presence of NADPH and O2. In addition evidence is given for further transformation of demecolcine to colchicine in the presence of acetyl-CoA and NADPH.

Alternative final steps in berberine biosynthesis in Coptis japonica cell cultures.

In Coptis japonica cell cultures an alternative pathway has been discovered which leads from (S)-tetrahydrocolumbamine via (S)-canadine to berberine. The two enzymes involved have been partially purified. (S)-Tetrahydrocolumbamine is stereospecifically transformed into (S)-canadine under formation of the methylenedioxy bridge in ring A. This new enzyme was named (S)-canadine synthase. (S)-Canadine in turn is stereospecifically dehydrogenated to berberine by an oxidase, (S)-canadine oxidase (COX), which was partially purified (25-fold). This enzyme has many physical properties in common with the already known (S)-tetrahydroprotoberberine oxidase from Berberis but grossly differs from the latter enzyme in its cofactor requirement (Fe) and its substrate specificity. Neither (S)-norreticuline nor (S)-scoulerine serves as substrate for the Coptis enzyme, while both substrates are readily oxidized by the Berberis enzyme. The four terminal enzymes catalyzing the pathway from (S)-reticuline to berberine are housed in Berberis as well as in Coptis in smooth vesicles with a density of ρ=1.14 g/ml. These vesicles have been enriched and characterized by electron microscopy.

S-Adenosyl-L-methionine: Columbamine-O-methyl transferase, a compartmentalized enzyme in protoberberine biosynthesis.

Suspension cultures of Berberis wilsoniae var. subcaulialata and B. aggregata are a useful source for the detection, partial purification and characterization of a new enzyme which specifically transfers the methyl group from (S)-adenosyl-L-methionine to the 2-OH-position of columbamine, thus producing palmatine. The enzyme was enriched approx. 30-fold; it is situated in a vesicle with the density ρ=1.14, has a pH-optimum of 8.9, a molecular weight of 52 kD and shows a high degree of substrate specificity.

S-adenosyl-L-methionine: (S)-scoulerine 9-O-methyltransferase, a highly stereo- and regio-specific enzyme in tetrahydroprotoberberine biosynthesis.

Suspension cultures of Berberis species are useful sources for the detection and isolation of a new enzyme which transfers the methyl group from S-adenosyl-L-methionine specifically to the 9-position of the (S)-enantiomer of scoulerine, producing (S)-tetrahydrocolumbamine. The enzyme was enriched 27-fold; it is not particle bound, has a pH optimum of 8.9, a molecular weight of 63 000 and shows a high degree of substrate specificity.

A Highly Specific O-Methyltransferase for Nororientaline Synthesis Isolated from Argemone platyceras Cell Cultures.

A highly specific new enzyme, S-adenosyl-L-methionine: (6-O-methyl-norlaudanosoline)-5'-O-methyltransferase which catalyses the formation of nor-orientaline from 6-O-methyl-norlaudanosoline and SAM was discovered, partially purified, and characterized. ARGEMONE PLATYCERAS cell suspension cultures served as enzyme source.

Partial Purification and Properties of S-Adenosylmethionine: (R), (S)-Norlaudanosoline-6-O-Methyltransferase from Argemone platyceras Cell Cultures.

A new enzyme, S-adenosylmethionine: (R), (S)-norlaudanosoline-6-O-methyltransferase, was isolated from the soluble protein extract of A. PLATYCERAS cell cultures and purified approximately 80-fold. This enzyme catalyses the formation of 6-O-methylnorlaudanosoline, and, to a minor extent, 7-O-methylnorlaudanosoline from SAM and (S), as well as (R), norlaudanosoline. The apparent molcular weight of the enzyme is 47000 Dalton. The pH-optimum of the enzyme is 7.5, the temperature optimum, 35 degrees C. Apparent K (M) values for (S) and (R)-norlaudanosoline were 0.2 mM, and for SAM, 0.05 mM. The transferase shows high substrate specificity for tetrahydrobenzylisoquinoline alkaloids. Simple orthophenols, like phenylpropane derivatives, coumarins or flavonoids, are not accepted as substrates. The enzyme is widely distributed in benzylisoquinoline-containing plant cell cultures and is present in differentiated plants like PAPAVER SOMNIFERUM.