(S)-tetrahydroprotoberberine oxidase the final enzyme in protoberberine biosynthesis

Tetrahedron Letters1Vol.25,No.9,pp Printed in Great Britain

(S)-TETRAHYDROPROTOBERBERINE

OXIDASE

IN PROTOBERBERINE N. Nagakura+,

Lehrstuhl

Pharmazeutische

THE FINAL

ENZYME

BIOSYNTHESIS

and M.H.

M. Amann,

0040-4039/84 $3.00 + .OO 01984 Pergamon Press Ltd.

953-954,1984

Zenk

Biologie,

Universitat

Miinchen,

8000 Miinchen 2, W-Germany +

Kobe Women's

Abstract:

College

A new

of Pharmacy,

enzyme

flavin

of O2 catalyzes

the presence

protoberberines

Kobe

658, Japan

has been discovered

the oxidation

to protoberberines

of

which

in

(S)-tetrahydro-

via the intermediate

7,14-

dehydroberberinium.

As a result

of previous

in the biosynthesis

it has been proposed

studies,

of the berberine

alkaloids

catalyzes

berberines assayed

to the corresponding

for its catalytic

sorption berine

the dehydrogenation

or by measuring

the dehydrogenation occur

in a number

beris

which

whole

plants

beris

wilsonae

of tritium

amounts

subcauZiaZata

described

contains catalytic

(excitation

at 450 nm, max.

morine

(50% inhibition

protoberberines tested

acted

I,3 PM and

at 0,18

of the

for

The catalytic

PM). The enzyme

The KM value

(S)-canadine

influences mole

the catalytic

of H202

Addition

reduces

is specific

for

1,5 Mole

by each mole the oxygen 953

in

from Bergrown

factor

150;

of 100.000,

a yellow-green

520 nm) which

is

is inhibited

by

for tetrahydro(R)-enantiomers

(S)-tetrahydrojatrorrhizine

is 26,7 PM. The aromatic

activity.

and H20 produced

of catalase

activity

to

of Ber-

were

weight

and none of the

rings A and D of the tetrahydroprotoberberine

which

(purification

spectrum

during

found

but also

at 8,9 and shows

emission

was

isolated

has a molecular

(S)-configuration,

as substrates.

first

cell cultures,

activity

of Na2S204.

was

in ab-

to ber-

straines

of protoberberines2

flavin,

for maximal

by the addition

those

was

medium

The enzyme

to homogeneity

fluorescence

the increase of canadine

especially

Schneid.

and purified

The enzyme

into the aqueous

The enzyme

somniferum.

a pH-optimum

quenched

by following

the oxidation

cultures,

considerable

of Papaver

25%). The enzyme

yield:

cell

(S)-tetrahydroproto-

alkaloids.

[8 ,I 3 ,l 4-3H3]-(R,S)-canadine.

of plant

var.

either

during

the release

of

produce

as previously

activity

step oxid-

here on a new enzyme

of some 20 different

protoberberine

(log E: 4,40)

at 345 nm

in the specific

We report

ation of ring C of tetrahydroprotoberberines'. which

that the final

consists

substitution

nucleus of oxygen

is consumed

of substrate

consumption

pattern

only quantitatively

which

and one

is oxidized.

rate to 67% of the

is of

954

original

value.

to berberine rium which

In the presence

but reisolated is present

of NaBD4

canadine

at the C-14

carbon

hydrocanadine

and canadine

methosalt

substitution

at C-8 or C-13

renders

able

for enzymatic

catalyzes

C-14 and N-7 further

is exclusively

from the cells

using

organelles

mitochondria,

(by mass

It is therefore

and Fonds

density

Supported

gradient

by SFB

to known

2. Hinz,

H., and M.H.

molecule

which

(Received

in Cermany

in scheme

to Alkaloids.

(p = 1,14 g/cm3).

particles

such

as

Forschungsgemeinschaft,

67,

19831

John Wily 620

(1981).

1.

can be isolated

etc.

145 of Deutsche

19 December

at

aromatizes

as shown

Industrie.

Zenk. Naturwiss.

Methyl unsuit-

References: 1. Cordell, G.A.: Introduction New York. 1981.

1X,14-De-

that the enzyme

centrifugation

in density

proplastids

der Chemischen

spectra).

intermediate molecule

canadine

atom of deute-

by the enzyme.

concluded

in a type of organelle

correspond

1

tetrahydroprotoberberines

the protoberberine

sucrose

do not

and NMR

are not oxidized these

of

of the tetrahydroprotoberberine

found

microbodies,

Acknowledgement: Bonn,

C to yield

does not oxidize

incorporation

and that the 7,14_dehydroberbinium

in ring

The enzyme

These

oxidation.

the dehydrogenation

the enzyme

shows

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