A microsomal cytochrome P-450-NADPH dependent enzyme which hydroxylates stereo- and regiospecifically carbon atom 14 of (S)- -N- methyltetrahydroprotoberberines has been discovered in a number of plant cell cultures originating from species containing protopine alkaloids; the monooxygenase was solubilized, partially purified (100-fold) and characterized

Battersby

Diesperger

Higashi

M. Rueffer

M.H. Zenk

Preininger

Rueffer

Rönsch

Schumacher

Takao

Tani

English

Rüffer, Martina

Zenk, Meinhart H.

Universität München: Elektronischen Publikationen

Tetrahedron Letters,Vo1.28,No.44,pp 5307-5310,1987 
Printed in Great Britain 
oo40-4039/87 $3.00 + .oo 
Pergamon Journals Ltd. 
ENZYMATIC FORMATION OF PROTOPINES BY A MICROSOMAL 
CYTOCHROME P-450 SYSTEM OF CORYDALIS VAGINANS 
M. Rueffer* and M.H. Zenk 
Lehrstuhl fiir Pharmazeutische Biologie, Universitat 
Munchen, Karlstr. 29, D-8000 Milnchen 2, W-Germany 
Abstract: A microsomal cytochrome P-450-NADPH 
dependent enzyme which hydroxylates stereo- and 
regiospecifically carbon atom 14 of (S)-cis-N- 
methyltetrahydroprotoberberines has been discover- 
ed in a number of plant cell cultures originating 
from species containing protopine alkaloids; the 
monooxygenase was solubilized, partially purified 
(lOO-fold) and characterized. 
The protopines are one of the most widely distributed groups of alka- 
loids in the plant kingdom [II, characterized by the presence of a ketone 
group at C-14. Feeding experiments with differentiated plants and callus 
cultures using 14 C- [21 or I3 C-labelled 131 tetrahydroprotoberberines 
or their N-methyl derivatives have shown that these compounds are the 
immediate precursors of protopines. During the oxygenation process it was 
demonstrated that the hydrogen atom at C-14 of a tetrahydroprotoberberine 
such as stylopine was completely lost 121. The fact that N-methylation of 
tetrahydroprotoberberines occurs prior to oxygenation was clearly shown by 
in vivo 131 feeding and enzyme L41 experiments. As part of our investigat- -- 
ion involving the elucidation of reaction sequences in isoquinoline bio- 
synthesis, we searched for the enzyme which catalyzes the hydroxylation of 
N-methyltetrahydroprotoberberines. 
A number of cytochrome P-450 dependent hydroxylating systems have been 
previously found to occur in microsomal preparations of higher plants as 
reviewed in C51. Microsomes from 4-day-old cultured suspension cells of 
Corydalis vaginans were prepared by standard methods C61. The MgC12 pelleted 
microsomal fraction was resuspended in 0.1 M tricine buffer pH 8.5 contain- 
ing 50 mM MgC12 and 5 mM thioglycolic acid. The protein (10 - 100 ug) was 
5307 
5308 
incubated in the presence of 200 mM tricine buffer pH 8.5, 200 uM NADPH and 
2 uM (S)-[8,14-3H1-~-N-methylcanadine or the correspondingly labelled 
stylopine (30 000 dpm; prepared enzymatically T.41) in a total volume of 
250 ul for 20 min at 30°C. The enzyme reaction was monitored by measuring 
the release of tritium into the aqueous phase of the incubation mixture 
[71. As a control assays with heat denatured protein were used. Enzym- 
atically catalyzed removal of tritium was observed in microsomal prepar- 
ations from cell suspension cultures of the Fumariaceae and Papaveraceae 
(Table I) but neither in those of the Berberidaceae or Ranunculaceae nor in 
cell cultures which do not produce isoquinoline alkaloids like Catharanthus 
roseus or Nicotiana tabacum. 
TABLE I. Survey of distribution of hydroxylation activity in different 
cell cultures 
Plant family Cell culture Enzyme activity 
(pkat/g dry weight) 
Fumariaceae 
Papaveraceae 
Corydalis ophiocarpa 52 
Corydalis vaginans 52 
Dicentra spectabilis 79 
Fumaria capreolata 84 
Fumaria officinalis 260 
Fumaria parviflora 137 
Bocconia cordata 117 
Eschscholtzia californica 73 
Glaucium flavum 69 
Papaver somniferum 44 
The product of the reaction was unequivocally identified as the proto- 
pine molecule, allocryptopine, by a large scale (600-fold) incubation 
containing 60 mg protein from C. vaginans in the presence of (S)-cis-N- - 
methylcanadine. The incubation mixture was freeze dried, chromatographed 
(TLC), and the newly formed compound isolated. The mass spectrum of the 
unknown product showed m/e M+ at 369 (4.6%); 341 (4.6%); 297 (4.6%); 268 
(16.3%); 206 (27.9%); 164 (100%); 163 (20.9%); 149 (16.3%). The mass 
spectrum was in every aspect identical to that of allocryptopine. Exactly 
the theoretical value of 33% of the radioactivity of the substrate molecu le 
5309 
was removed during this transition. The enzyme catalyzes the hydroxylation 
of the (S)-cis-N-methylderivatives of canadine (lOO%), stylopine (80%), 
tetrahydrothalifendine (30%), tetrahydropalmatine (19%), and corydalmine 
(6%). The (R)-cisltrans-N-methylcanadine epimeric mixture was completely -- 
inactive which fact indicated that synthetic (R,S)-cis/trans-N-methyltetra- -- 
hydroprotoberberines can be interchangeably used with (S)-=-N-methyl- 
tetrahydroprotoberberines in subsequent work. 
The enzyme complex was solubilized from microsomal membranes by sodium 
cholate and the proteins purified further on an affinity column containing 
Procion red H3B as the ligand. In this manner, the enzyme complex was 
purified 100-fold with a 35% overall yield. The final preparation had a 
specific activity of 425 pkat/mg protein. The optimum of the reaction was 
at pH 8.5 and 30°C with half maximal activity at pH 6.5 and 9.7, as well as 
10 and 45°C. In the absence of molecular oxygen no reaction was observed. 
Replacement of NADPH with NADH resulted in only 33% of the original activ- 
ity. NAD and NADP were both ineffective. The KM was 12.5 pM for cis-N- 
methylcanadine and 62.5 uM for NADPH. The UV spectrum of the Na2S204 
reduced enzyme (tricine buffer pH 8.5) showed peaks at 420, 450 and 480 nm 
suggesting a cytochrome P-450 enzyme. Characteristic absorption changes 
occurred in the presence of carbon monoxide which, in spite of the presence 
of 02' rendered the enzyme inactive. The carbon monoxide inhibition was 
reversed in light. The enzyme complex was inhibited neither by CN- nor by 
EDTA. The concentration for 50% inhibition of the enzyme activity was 
determined for the typical inhibitors of cytochrome P-450: ketoconazole 
(1.5 uM), metyrapone (2 uM), prochloraz (6 uM), ancymidol (12 uM), and 
cytochrome c (40 uM). Collectively, these properties of the enzyme clearly 
show that the hydroxylase is a cytochrome P-450 containing enzyme complex. 
The enzyme responsible for the formation of this important metabolite is 
termed: (S)-cis-N-methyltetrahydroprotoberberine-l4-hydroxylase. It is the 
first monooxygenase discovered in benzylisoquinoline alkaloid metabolism. 
The protopine alkaloids, of which about twenty differently substituted 
members are known, arise biosynthetically by action of these hydroxylases 
with possibly differing substrate specificities. Protopine alkaloids can be 
further converted to benzazepine and benzophenanthridine alkaloids as has 
been shown by in vivo experiments [3,8-101. It is our continued interest to -- 
clarify the biosynthesis of these alkaloids at the enzyme level. 
5310 
NADPH, 02 
* 
OR1 
OR2 
R1 = R2 = CH3 : (S)-N-methylcanadine 
RI + Rz =-CH2-: (S)-N-methylstylopine. 
OR1 
OR2 
OR1 
OR2 
R1 = R2 = CH3 : allocryptopine 
R1 + Rg =-CH2-: protopine 
Acknowledgement: 
Our thanks are due to Ms. S. Kunz for excellent technical assistance and 
Dr. T.M. Kutchan for linguistic help in preparation of the manuscript. 
Supported by SFB 145 of Deutsche Forschungsgemeinschaft, Bonn, and Fends 
der Chemischen Industrie. 
References 
1. V. Preininger. In: The Alkaloids (A. Brossi ed.). Vol. 29, 1. - 
Academic Press New York - London (1986). 
2. A.R. Battersby, J. Staunton, H.R. Wiltshire, R.J. Francis, and 
R. Southgate. J.Chem.Soc. Perkin I, 1147 (1975). 
3. N. Takao, M. Kamigauchi and M. Okada. Helv.Chim.Acta 66, 473 (1983). 
4. M. Rueffer and M.H. Zenk. Tetrahedron Letters 27, 5603 (1986). 
5. K. Higashi. GANN Monograph on Cancer Research 30, 49 (1985). - 
6. H. Diesperger, C.R. Muller and H. Sandermann. FEBS Letters 43, 
155 (1974). 
7. H.-M. Schumacher, M. Rueffer, N. Nagakura, and M.H. Zenk. Planta 
medica 48, 212 (1983). - 
8. H. Ronsch. Eur.J.Biochem. 28, 123 (1972). - 
9. H. Ronsch. Phytochem. 2, 691 (1977). 
10. C. Tani and K. Tagahara. Chem.Pharm.Bull. 22, 2457 (1974). 
(Received in Germany 24 July 1987) 


Enzymatic formation of protopines by a microsomal cytochrome P-450 system of Corydalis vaginans

Open Access LMU

Tetrahedron Letters,Vo1.28,No.44,pp 5307-5310,1987 Printed in Great Britain oo40-4039/87 $3.00 + .oo Pergamon Journals Ltd. ENZYMATIC FORMATION OF PROTOPINES BY A MICROSOMAL CYTOCHROME P-450 SYSTEM OF CORYDALIS VAGINANS M. Rueffer* and M.H. Zenk Lehrstuhl fiir Pharmazeutische Biologie, Universitat Munchen, Karlstr. 29, D-8000 Milnchen 2, W-Germany Abstract: A microsomal cytochrome P-450-NADPH dependent enzyme which hydroxylates stereo- and regiospecifically carbon atom 14 of (S)-cis-N- methyltetrahydroprotoberberines has been discover- ed in a number of plant cell cultures originating from species containing protopine alkaloids; the monooxygenase was solubilized, partially purified (lOO-fold) and characterized. The protopines are one of the most widely distributed groups of alka- loids in the plant kingdom [II, characterized by the presence of a ketone group at C-14. Feeding experiments with differentiated plants and callus cultures using 14 C- [21 or I3 C-labelled 131 tetrahydroprotoberberines or their N-methyl derivatives have shown that these compounds are the immediate precursors of protopines. During the oxygenation process it was demonstrated that the hydrogen atom at C-14 of a tetrahydroprotoberberine such as stylopine was completely lost 121. The fact that N-methylation of tetrahydroprotoberberines occurs prior to oxygenation was clearly shown by in vivo 131 feeding and enzyme L41 experiments. As part of our investigat- -- ion involving the elucidation of reaction sequences in isoquinoline bio- synthesis, we searched for the enzyme which catalyzes the hydroxylation of N-methyltetrahydroprotoberberines. A number of cytochrome P-450 dependent hydroxylating systems have been previously found to occur in microsomal preparations of higher plants as reviewed in C51. Microsomes from 4-day-old cultured suspension cells of Corydalis vaginans were prepared by standard methods C61. The MgC12 pelleted microsomal fraction was resuspended in 0.1 M tricine buffer pH 8.5 contain- ing 50 mM MgC12 and 5 mM thioglycolic acid. The protein (10 - 100 ug) was 5307 5308 incubated in the presence of 200 mM tricine buffer pH 8.5, 200 uM NADPH and 2 uM (S)-[8,14-3H1-~-N-methylcanadine or the correspondingly labelled stylopine (30 000 dpm; prepared enzymatically T.41) in a total volume of 250 ul for 20 min at 30°C. The enzyme reaction was monitored by measuring the release of tritium into the aqueous phase of the incubation mixture [71. As a control assays with heat denatured protein were used. Enzym- atically catalyzed removal of tritium was observed in microsomal prepar- ations from cell suspension cultures of the Fumariaceae and Papaveraceae (Table I) but neither in those of the Berberidaceae or Ranunculaceae nor in cell cultures which do not produce isoquinoline alkaloids like Catharanthus roseus or Nicotiana tabacum. TABLE I. Survey of distribution of hydroxylation activity in different cell cultures Plant family Cell culture Enzyme activity (pkat/g dry weight) Fumariaceae Papaveraceae Corydalis ophiocarpa 52 Corydalis vaginans 52 Dicentra spectabilis 79 Fumaria capreolata 84 Fumaria officinalis 260 Fumaria parviflora 137 Bocconia cordata 117 Eschscholtzia californica 73 Glaucium flavum 69 Papaver somniferum 44 The product of the reaction was unequivocally identified as the proto- pine molecule, allocryptopine, by a large scale (600-fold) incubation containing 60 mg protein from C. vaginans in the presence of (S)-cis-N- - methylcanadine. The incubation mixture was freeze dried, chromatographed (TLC), and the newly formed compound isolated. The mass spectrum of the unknown product showed m/e M+ at 369 (4.6%); 341 (4.6%); 297 (4.6%); 268 (16.3%); 206 (27.9%); 164 (100%); 163 (20.9%); 149 (16.3%). The mass spectrum was in every aspect identical to that of allocryptopine. Exactly the theoretical value of 33% of the radioactivity of the substrate molecu le 5309 was removed during this transition. The enzyme catalyzes the hydroxylation of the (S)-cis-N-methylderivatives of canadine (lOO%), stylopine (80%), tetrahydrothalifendine (30%), tetrahydropalmatine (19%), and corydalmine (6%). The (R)-cisltrans-N-methylcanadine epimeric mixture was completely -- inactive which fact indicated that synthetic (R,S)-cis/trans-N-methyltetra- -- hydroprotoberberines can be interchangeably used with (S)-=-N-methyl- tetrahydroprotoberberines in subsequent work. The enzyme complex was solubilized from microsomal membranes by sodium cholate and the proteins purified further on an affinity column containing Procion red H3B as the ligand. In this manner, the enzyme complex was purified 100-fold with a 35% overall yield. The final preparation had a specific activity of 425 pkat/mg protein. The optimum of the reaction was at pH 8.5 and 30°C with half maximal activity at pH 6.5 and 9.7, as well as 10 and 45°C. In the absence of molecular oxygen no reaction was observed. Replacement of NADPH with NADH resulted in only 33% of the original activ- ity. NAD and NADP were both ineffective. The KM was 12.5 pM for cis-N- methylcanadine and 62.5 uM for NADPH. The UV spectrum of the Na2S204 reduced enzyme (tricine buffer pH 8.5) showed peaks at 420, 450 and 480 nm suggesting a cytochrome P-450 enzyme. Characteristic absorption changes occurred in the presence of carbon monoxide which, in spite of the presence of 02' rendered the enzyme inactive. The carbon monoxide inhibition was reversed in light. The enzyme complex was inhibited neither by CN- nor by EDTA. The concentration for 50% inhibition of the enzyme activity was determined for the typical inhibitors of cytochrome P-450: ketoconazole (1.5 uM), metyrapone (2 uM), prochloraz (6 uM), ancymidol (12 uM), and cytochrome c (40 uM). Collectively, these properties of the enzyme clearly show that the hydroxylase is a cytochrome P-450 containing enzyme complex. The enzyme responsible for the formation of this important metabolite is termed: (S)-cis-N-methyltetrahydroprotoberberine-l4-hydroxylase. It is the first monooxygenase discovered in benzylisoquinoline alkaloid metabolism. The protopine alkaloids, of which about twenty differently substituted members are known, arise biosynthetically by action of these hydroxylases with possibly differing substrate specificities. Protopine alkaloids can be further converted to benzazepine and benzophenanthridine alkaloids as has been shown by in vivo experiments [3,8-101. It is our continued interest to -- clarify the biosynthesis of these alkaloids at the enzyme level. 5310 NADPH, 02 * OR1 OR2 R1 = R2 = CH3 : (S)-N-methylcanadine RI + Rz =-CH2-: (S)-N-methylstylopine. OR1 OR2 OR1 OR2 R1 = R2 = CH3 : allocryptopine R1 + Rg =-CH2-: protopine Acknowledgement: Our thanks are due to Ms. S. Kunz for excellent technical assistance and Dr. T.M. Kutchan for linguistic help in preparation of the manuscript. Supported by SFB 145 of Deutsche Forschungsgemeinschaft, Bonn, and Fends der Chemischen Industrie. References 1. V. Preininger. In: The Alkaloids (A. Brossi ed.). Vol. 29, 1. - Academic Press New York - London (1986). 2. A.R. Battersby, J. Staunton, H.R. Wiltshire, R.J. Francis, and R. Southgate. J.Chem.Soc. Perkin I, 1147 (1975). 3. N. Takao, M. Kamigauchi and M. Okada. Helv.Chim.Acta 66, 473 (1983). 4. M. Rueffer and M.H. Zenk. Tetrahedron Letters 27, 5603 (1986). 5. K. Higashi. GANN Monograph on Cancer Research 30, 49 (1985). - 6. H. Diesperger, C.R. Muller and H. Sandermann. FEBS Letters 43, 155 (1974). 7. H.-M. Schumacher, M. Rueffer, N. Nagakura, and M.H. Zenk. Planta medica 48, 212 (1983). - 8. H. Ronsch. Eur.J.Biochem. 28, 123 (1972). - 9. H. Ronsch. Phytochem. 2, 691 (1977). 10. C. Tani and K. Tagahara. Chem.Pharm.Bull. 22, 2457 (1974). (Received in Germany 24 July 1987) 

Enzymatic formation of protopines by a microsomal cytochrome P-450 system of Corydalis vaginans

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