(Received for publication, June 20, 1995; and in revised form, July 25, 1995)
From the
The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine. This is a reaction that has neither an equivalent in organic chemistry nor a parallel in nature. The uniqueness of this catalytic reaction prompted an in depth study that began with the isolation of the cDNA encoding the berberine bridge enzyme followed by the overexpression of this cDNA in insect cell culture. The heterologously expressed enzyme has herein been shown to contain covalently attached FAD in a molar ratio of cofactor to protein of 1:1.03. Site-directed mutagenesis and laser desorption time-of-flight mass spectrometry suggest that the site of covalent attachment is at His-104. The holoenzyme exhibited absorbance maxima at 380 and 442 nm and a fluorescence emission maximum at 628 nm (310 nm excitation). Enzymic transformation of a series of (S)-reticuline derivatives modified with respect to the stereochemistry at C-1 or in the aromatic ring substitution suggests that ring closure proceeds in two steps: formation of the methylene iminium ion and subsequent ring closure via an ionic mechanism.
The berberine bridge enzyme ((S)-reticuline:oxygen oxidoreductase (methylene-bridge-forming), EC 1.5.3.9) transforms the N-methyl group of (S)-reticuline into the berberine bridge carbon, C-8, of (S)-scoulerine, thereby forming the protoberberine carbon skeleton(1, 2) . (S)-Scoulerine then serves as the biosynthetic precursor to a multitude of species-specific protopine, protoberberine and benzophenanthridine alkaloids (Fig. 1). The interest in these alkaloid families stems from their pharmacological activities. Berberine, for example, is currently used as an antibacterial treatment for eye infections in Europe and for intestinal infections in the Far East. The benzophenanthridine alkaloid sanguinarine is an antimicrobial used in the treatment of peridontal disease in both the United States and Europe.
Figure 1: Reaction catalyzed by the berberine bridge enzyme. The berberine bridge enzyme, BBE, catalyzes the oxidative cyclization of the N-methyl moiety of (S)-reticuline to the berberine bridge carbon C-8 of (S)-scoulerine that is the precursor to a broad spectrum of isoquinoline alkaloids including the protoberberines, protopines, and benzophenanthridines. These alkaloid classes are widely distributed in plant families such as the Berberidaceae, Fumariaceae, and Papaveraceae and contain many members that are pharmacologically active.
The benzophenanthridine alkaloids also have an important
function in the plant. It was shown with cell suspension cultures of Eschscholtzia californica, the California poppy, that these
antimicrobial alkaloids (3, 4) accumulate in response
to the addition of elicitors to the culture medium(5) . Five of
the seven biosynthetic enzymes that lead from (S)-reticuline
to sanguinarine are induced in response to this elicitor
treatment(6) . The cDNA for one of these inducible enzymes, the
berberine bridge enzyme, has been isolated and used as a probe in RNA
gel blot experiments to demonstrate that the gene bbe1 is
transcribed prior to accumulation of the enzyme in the cell
culture(7) . Each of these five inducible enzymes is
membrane-associated. The berberine bridge enzyme, in particular, is
accumulated within a subcellular particle of a density =
1.14 g/ml(8, 9) .
With respect to the mechanism of
the berberine bridge enzyme, the closure of ring C in the conversion of (S)-reticuline to (S)-scoulerine was demonstrated to
arise by an oxidative cyclization of the N-methyl group (10, 11) and is a reaction that is not currently
chemically achievable. There are also no other known examples of this
type of reaction in species that do not produce protoberberine
alkaloids. The mechanism with which the berberine bridge enzyme
directly converts the N-methyl moiety into the berberine
bridge carbon is of high interest to elucidate. Characterization of
this enzyme has been limited by the quantity of homogeneous enzyme that
could be isolated from plant cell cultures. The original
characterization was performed on enzyme purified from Berberis
beaniana cell suspension cultures(2) . In that scheme, 11
µg of pure protein were obtained after an eight-step purification
procedure. The homogeneous Berberis enzyme was characterized
as a single polypeptide with a relative molecular mass of 52 ± 4
kDa. In the presence of oxygen, the enzyme converts (S)-reticuline, (S)-protosinomenine, and (S)-laudanosoline to the corresponding (S)-tetrahydroprotoberberines with a stoichiometric release of
HO
. Inhibition by chelating agents suggested
the involvement of a metal ion in catalysis. With the limited quantity
of Berberis enzyme available, the formation of the berberine
bridge carbon from (S)-[N-methyl-
H
,
H]reticuline
(chiral methyl (S)-reticuline) was shown by
H NMR
to proceed with an inversion of configuration(12) .
A more
thorough biochemical characterization of the enzyme that catalyzes this
unique reaction requires large quantities of pure protein. The
berberine bridge enzyme cDNA was, therefore, expressed in insect cell
culture in a baculovirus vector(13) . The overexpression
resulted in 4 mg of pure active enzyme/liter of cell culture that
showed a K value for (S)-reticuline and pH and temperature optima identical to that
of the native Eschscholtzia enzyme. The enzyme was readily
purified in a facile, three-step procedure. In order to obtain these
same yields from Eschscholtzia, over 300 liters of elicited
cell suspension cultures would have to be extracted. It is the pure,
heterologously expressed enzyme that is used in the characterization
experiments described herein.
The structure of the products was identifed by repeating the
nonradioactive assay and applying the entire sample onto a 0.25-mm
silica gel 60 F thin-layer chromatography plate (Merck)
and resolving the components with methylene chloride, methanol, 25%
ammonium hydroxide (90:9:1) or chloroform, acetone, diethylamine
(50:40:10). The product band was scraped from the plate, eluted with
methanol, dried in vacuo, and redissolved in a minimal volume
of methanol prior to mass spectral analysis on a Finnigan MAT
quadrupole SSQ 700 mass spectrometer. When a dehydrogenation reaction
was suspected, the eluted product was first reduced with
NaB
H
in methanol, made to 500 µl with 20
mM potassium phosphate buffer, pH 9.3, and extracted 5 times
with 300 µl of ethyl acetate. The ethyl acetate was removed in
vacuo, and the sample was redissolved in a minimal volume of
methanol for mass spectral analysis. Duplicate samples were reduced
with NaBH
for comparison.
The ratio of tritium
released was determined by performing the same enzyme assay as above,
but unlabeled formaldehyde was not added. Instead, the radioactive
formaldehyde was trapped by the addition of 720 µg of dimedone in
80 µl of 50% methanol. The reaction was allowed to proceed
overnight at room temperature. One-half of the formaldimethone solution
was reacted with activated charcoal for 72 h to absorb the tritiated
formaldimethone. The charcoal was removed by centrifugation for 5 min
at room temperature in an Eppendorf microcentrifuge. The radioactivity
in the supernatant (HOOH and HO
H) as well as
that in the sample not treated with charcoal (tritiated formaldimethone
plus HOO
H and HO
H) was determined by liquid
scintillation counting. All values were corrected for recovery of
radioactivity.
The concentrated (6 mg/ml) enzyme was deeply yellow colored. Due to the absence of iron, a flavin cofactor was suspected. In support of the presence of a flavin cofactor, a comparison of the translated nucleotide sequence of the berberine bridge enzyme cDNA clone (7) demonstrated 25% identity to 6-hydroxy-D-nicotine oxidase from Arthrobacter oxidans and that it retained the peptide sequence known to contain the His residue to which the FAD in 6-hydroxy-D-nicotine oxidase is attached(17, 18) . Analysis for autofluorescence of pure berberine bridge enzyme by SDS-polyacrylamide gel electrophoresis yielded a single yellow-green protein band that could be visualized in the presence of 7% acetic acid by transillumination at 366 nm (Fig. 2). This autofluorescence was further characterized, and the fluorescence emission spectrum that was obtained (Fig. 3A) is typical of that for a flavoenzyme. The fluorescence emission could be completely quenched by the addition of dithionite to the enzyme solution. The absorbance spectrum of the berberine bridge enzyme shows maxima at 380 and 442 nm that are also indicative of flavin (Fig. 3B). The molar ratio of flavin to protein was spectrophotometrically determined to be 1:1.03.
Figure 2: Berberine bridge enzyme analyzed by SDS-polyacrylamide gel electrophoresis. PanelA displays an SDS-polyacrylamide gel electrophoresis gel containing 10 and 30 µg of pure berberine bridge enzyme (arrow). The protein bands were visualized with Coomassie Brilliant Blue R-250. PanelB shows the same gel as in A with the protein bands visualized by transillumination at 366 nm.
Figure 3: Spectral measurements of the berberine bridge enzyme. PanelA, the enzyme (2 mg) dissolved in 3 ml of 20 mM potassium phosphate buffer, pH 7.5, was irradiated at 310 nm, and the fluorescence emission maximum was determined to be at 628 nm. The fluorescence was quenched by the addition of sodium dithionite. PanelB, the absorption spectrum of enzyme (1 mg) dissolved in 1 ml of 20 mM potassium phosphate buffer, pH 7.5, resulted in two maxima at 380 and 442 nm. BBE, berberine bridge enzyme.
Since the berberine bridge enzyme contained a consensus
sequence RSGGHSYEGLS (amino acid positions 100-110) for the
covalent attachment of a flavin cofactor and since boiling of the
enzyme in a denaturing buffer for SDS-polyacrylamide gel
electrophoresis analysis failed to destroy the autofluorescence of the
enzyme in the gel, it was suspected that a flavin was covalently
attached to the His-104 residue within the consensus sequence. To test
this possibility, several His residues as well as an Arg residue, which
is thought to be involved in the autoflavinylation of
6-hydroxy-D-nicotine oxidase (19) , were altered by
site-directed mutagenesis. The change of His-39 to Gly and of His-408
to Ser did not destroy the ability of the enzyme to convert (S)-reticuline to (S)-scoulerine (40 and 5%
conversion, respectively), but the change of His-104 to Thr and of
Arg-100 to Thr each completely abolished enzyme activity, indicating
that the latter two residues might be necessary for covalent attachment
of the cofactor. An attempt to quantitate the amount of each berberine
bridge enzyme mutant produced by a Western blot incubated with
polyclonal antibodies raised against heterologously expressed berberine
bridge enzyme was unsuccessful. Only the wild-type enzyme and the
His-408 Ser mutant cross-reacted with the antibodies. Although
the His-39
Gly mutant was 40% as active as the wild-type enzyme,
it did not cross-react with the antibodies. Likewise, the enzymatically
inactive mutants His-104
Thr and Arg-100
Thr did not
cross-react with the berberine bridge enzyme antibodies. It must also
be considered here that the ratio of antigen to antibody could have
been changed due to instability of certain mutant enzymes and,
therefore, cross-reactivity was not observed.
In order to further
demonstrate that the site of covalent attachment could be His-104, the
enzyme was digested with S. aureus V8 endoproteinase Glu-C,
which hydrolyzes at the carboxyl-terminal end of glutamate and
aspartate amino acid residues, in an attempt to isolate the flavin
containing peptide from the protein. After digestion for 18 h, the
incubation mixture was centrifuged, and the intensely yellow pellet was
washed with water and then dissolved in acetonitrile. Any attempts to
further purify this peptide by high performance liquid chromatography
were unsuccessful presumably due to its extremely hydrophobic nature.
The yellow peptide material was, therefore, directly analyzed by laser
desorption time-of-flight mass spectrometry and found to consist of two
major peptides, one with relative molecular mass 4634 and the other of
6248. A relative molecular mass of 6248 corresponds to the peptide
Leu-83 to Glu-133 plus FMN (theoretical relative molecular mass
-2HO = 6254) and lies within the accuracy of
this type of mass spectrometry. It cannot be excluded here that the
phosphodiester bond of FAD was fragmented during desorption of the
peptide, but this result does support His-104 as the site of covalent
flavin attachment.
The majority of covalently flavinylated enzymes
contain FAD, but there are exceptions such as trimethylamine
dehydrogenase from bacterium sp. WA
1 and
dimethylamine dehydrogenase from Hyphomicrobium X, in which
cases the FMN molecule is attached to the cysteinyl
sulfur(20) . In order to differentiate between FAD and FMN on
the berberine bridge enzyme, the enzyme was boiled for 10 min in 0.1 N HCl in order to hydrolyze the phosphodiester bond of FAD and
release AMP, and the yellow denatured protein was removed by
centrifugation. The supernatant was lyophillized, and the residue was
redissolved in ethanol and analyzed by mass spectrometry. For
comparison, two enzymes that are known to contain covalently bound FAD,
choline oxidase from A. globiformis and cholesterol oxidase
from Brevibacterium sp., were similarly treated and analyzed.
As a control, unhydrolyzed berberine bridge enzyme was lyophillized and
resuspended in ethanol, and the denatured protein was removed by
centrifugation. The ethanolic supernatant was also analyzed by mass
spectrometry. In the mass spectrum, hydrolyzed berberine bridge enzyme,
choline oxidase, and cholesterol oxidase contained ion fragments that
the unhydrolyzed berberine bridge enzyme preparation did not. In the
chemical ionization mode, a mass of m/z 122
(corresponding to adenine minus nitrogen) reproducibly appeared.
Likewise, with electron impact ionization, a fragment at m/z 43 (HN-CH=NH from the purine ring
system) was present in all three mass spectra but absent in the
control. The fragment at m/z 43 was also
characteristic of the standard adenine reference compound. A milder
hydrolysis of the enzymes with the phosphodiesterase Aspergillus
oryzae S1 nuclease yielded similar results.
Three tetrahydroprotoberberine alkaloids were also tested as
potential substrates for the enzyme (Table 2). While (S)-scoulerine, the natural enzymatic product, remained
unchanged, in both (S)-coreximine and (S)-norsteponine, a double bond between the nitrogen and C-8
was introduced. Both imines were reduced with either
NaBH
or NaBH
and analyzed by mass
spectrometry to determine the position of the double bond. Since these
types of molecules immediately fragment into the isoquinoline moiety
and the benzyl moiety in the mass spectrometer(21) , the
deuterium atom introduced by reduction was readily localized to C-8
when the mass spectrum (electron impact ionization, 70 eV) (m/z = 328, 58%; 178, 100%; 151, 50%; 136, 7%)
was compared with the same imine reduced with NaBH
(electron impact ionization, 70 eV) (m/z = 327, 100%; 178, 85%; 150, 58%; 135, 25%). (S)-Coreximine and (S)-norsteponine are positional
isomers that fragmented identically in the mass spectrometer.
Figure 4:
N-Demethylation of (S)-N-methylcoclaurine by the berberine bridge
enzyme. Proposed mechanism of N-demethylation derived from
formaldehyde trapping experiments with dimedone using (S)-N-[N-methyl-H]methylcoclaurine
as substrate. The first step, oxidation to the methylene iminium ion
proceeds enzymatically, while the second step, hydration of the double
bond and elimination of formaldehyde is most likely
spontaneous.
The ratio of tritium released in the N-demethylation reaction was determined by repeating the
formaldehyde trapping experiments but without the addition of unlabeled
formaldehyde. Starting with 600,000 cpm of (S)-N-[N-methyl-H]methylcoclaurine,
93% of the radioactivity was released during the demethylation
reaction. Of this 93% (559, 400 cpm), 363,600 cpm (65%) were recovered
as formaldimethone and 195,800 cpm (35%) were measured as tritiated
water (theoretical values 66 and 33%, respectively). Tritiated water
and tritiated hydrogen peroxide cannot be differentiated in this type
of experiment, but it is known from previous studies that the berberine
bridge enzyme produces 1 mol of H
O
for each mol
of (S)-reticuline converted(2) . Taken together, these
results suggest that the mechanism of N-demethylation of (S)-N-[N-methyl-
H]methylcoclaurine
by the berberine bridge enzyme is as depicted in Fig. 4in which
the enzyme oxidizes the substrate to the methylene iminium cation that
in turn spontaneously adds water and then eliminates formaldehyde.
In the previous studies of the berberine bridge enzyme mechanism of oxidative cyclization of (S)-reticuline to (S)-scoulerine, the quantity of pure enzyme was a limiting factor(2, 12) . A cofactor requirement could not be demonstrated, and the transformation of only small quantities of radioactive substances could be monitored. This problem has been overcome by the isolation and heterologous expression of the E. californica berberine bridge enzyme cDNA clone(7, 13) .
Several conclusions can be drawn from the results presented here. The berberine bridge enzyme is a flavinylated oxidase that contains one molecule of FAD covalently attached to His-104/molecule of protein. A consensus sequence at amino acid positions 100-110 derived from the translated nucleotide sequence of the cDNA clone contains the essential histidine residue and is consistent with those found in other known or suspected covalently flavinylated proteins such as A. oxidans 6-hydroxy-D-nicotine oxidase(18) , rat liver L-gulonolactone oxidase(22) , and a Streptomyces lavendulae mitomycin C resistance gene mcrA(23) . The complete amino acid sequence alignment of these four proteins is provided in (23) . Arg-100 of the berberine bridge enzyme is also essential to enzyme activity, but unlike for the equivalent Arg residue in 6-hydroxy-D-nicotine oxidase(19) , it could not be demonstrated here that it is essential for autoflavinylation, since reconstitution conditions for apoberberine bridge enzyme have not yet been successfully established.
The oxidative cyclization catalyzed by the berberine bridge enzyme can proceed through either an ionic or a radical intermediate(2, 7, 12) . In other words, the flavin can be reduced by either two one-electron transfers or one two-electron transfer from (S)-reticuline. In examples taken from other flavoproteins, a direct hydride transfer model is favored for the dehydrogenation of pyridine nucleotide by glutathione reductase, whereas monoamine oxidase is proposed to oxidize nonactivated amines by a radical mechanism although there is no strong evidence against a hydride transfer mechanism (for review, see (24) ). It has been difficult to reach absolute conclusions concerning the reaction mechanisms of flavoenzymes because flavins can accept and donate redox equivalents via a single hydride transfer, via two single-electron transfers, and can form covalent adducts with the substrate. There is also a lack of spectral evidence in the literature for flavin semiquinone radical intermediates.
To gain insight into the structural requirements of berberine bridge formation, 19 tetrahydrobenzylisoquinoline alkaloids were tested, and five of these were converted to protoberberines. The structural requirements for formation of the berberine bridge that can be concluded from these experiments are as follows. The N-methyltetrahydrobenzylisoquinoline nucleus must have the S-configuration at C-1, and the aromatic carbon ortho to position C-2` of ring closure must be substituted with a hydroxyl group. The substitution pattern of the aromatic ring moiety of the isoquinoline nucleus appears to be irrelevant since (S)-protosinomenine with the 6-hydroxy 7-methoxy substitution pattern and crassifoline with the 7-methoxy 8-hydroxy substitution pattern were both converted. This latter result has an implication in the biosynthesis of the protoberberine alkaloids clarkeanidine (25) and caseamine (26) that contain the crassifoline substitution pattern(27) . Since crassifoline is readily converted to the corresponding protoberberine, it can be expected that the berberine bridge enzyme found in plant species such as Corydalis(2) should be involved in the biosynthesis of both 6,7-substituted (reticuline-derived) and 7,8-substituted (crassifoline-derived) protoberberine alkaloids.
The oxidation of
the protoberberines (S)-coreximine and (S)-norsteponine to the corresponding N to C-8 iminium ion
suggested that the berberine bridge enzyme may first oxidize (S)-reticuline to the methylene iminium ion and then cyclize
via nucleophilic attack by C-2` of the phenyl ring. This is consistent
with the results of H NMR analysis of the stereochemical
course of ring closure of chiral methyl (S)-reticuline that
was found to proceed with an inversion of configuration(12) .
This was further corroborated by the oxidative N-demethylation
of (S)-N-methylcoclaurine by the enzyme. The
formaldehyde trapping experiments with dimedone demonstrate that the
berberine bridge enzyme oxidizes the N-CH
bond to the
methylene iminium ion that is then unable to form the berberine bridge
carbon due to the absence of a hydroxyl group on the phenyl ring that
would be ortho to the position of ring closure. This
intermediate is unstable and adds water, and formaldehyde is
spontaneously eliminated. The methylene iminium ion of (S)-reticuline can be synthetically prepared by reacting (S)-norreticuline with formaldehyde. The product cannot be
isolated, but it spontaneously cyclizes to a mixture of (S)-coreximine and (S)-scoulerine. The enzyme
catalyzed reaction, however, produces exclusively (S)-scoulerine, suggesting that ring closure is enzyme
mediated and that a hydroxyl moiety para to the position of
ring closure does not suffice for the enzymatic reaction. The phenyl
ring could either be held in a rigid position by the enzyme, or a
proton could be abstracted from the hydroxyl moiety to form the (S)-reticuline semiquinone only when it occupies the position ortho to ring closure due to proximity to a hypothetical basic
amino acid residue at the active site.
Unsuccessful attempts were made to detect by electron spin resonance spectroscopy a radical species during formation of the berberine bridge using 3 mg of enzyme and saturating concentrations of the substrate (S)-reticuline. No radical could be detected, and the product (S)-scoulerine precipitated. A search was then made for a substrate that might prove to be more suitable for electron spin resonance measurements. As a result, the inhibitor (S)-norreticuline was used in the next set of electron spin resonance measurements under the presumption that an electron could be abstracted from the nitrogen, but in the absence of the N-methyl group, the reaction could go no further, and the radical species would be stabilized by the aromatic ring system. Using again 3 mg of enzyme (a 100-fold excess of the minimum concentration necessary for detection of a radical species) and 10-100 µM (S)-norreticuline, no radical signal could be detected.
It can be concluded, however, that the oxidative cyclization of (S)-reticuline to (S)-scoulerine by the berberine bridge enzyme proceeds via a two-step mechanism (Fig. 5). The first step is oxidation to the methylene iminium ion. This is a reaction that is analogous to that catalyzed by amine oxidases. As for amine oxidases, it could occur by two one-electron removals, first from the nitrogen to form a radical cation and then from the methyl carbon to form the double bond, although a direct hydride abstraction from the N-methyl moiety cannot be excluded(24) . The second step is the stereospecific, ionic ring closure forming the berberine bridge of (S)-scoulerine.
Figure 5:
Proposed mechanism of berberine bridge
formation. The oxidative cyclization of (S)-reticuline to (S)-scoulerine proceeds via a two step mechanism: oxidation to
the methylene iminium ion followed by nucleophilic attack at the imine
carbon by C-2` to form C-8, the berberine bridge. B and B
are proposed basic
residues of the enzyme involved in semiquinone formation and
aromatization, respectively.