©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization and Mechanism of the Berberine Bridge Enzyme, a Covalently Flavinylated Oxidase of Benzophenanthridine Alkaloid Biosynthesis in Plants (*)

(Received for publication, June 20, 1995; and in revised form, July 25, 1995)

Toni M. Kutchan (§) Heinz Dittrich

From the Laboratorium für Molekulare Biologie, Universität München, Karlstrasse 29, 80333 München, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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 H(2)O(2). 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-^2H(1),^3H]reticuline (chiral methyl (S)-reticuline) was shown by ^3H 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.


EXPERIMENTAL PROCEDURES

Materials

The berberine bridge enzyme used in all experiments was expressed in Spodoptera frugiperda Sf9 cells and purified exactly according to (13) . All alkaloids were from the departmental collection. Biochemicals, NaBH(4) and NaB^2H(4), and choline oxidase from Arthrobacter globiformis and cholesterol oxidase from Brevibacterium sp. were obtained from Sigma (München); Staphylococcus aureus V8 endoproteinase Glu-C was from Boerhinger Mannheim. Plasmids pVL1393 and pWHA188 were from Invitrogen and Berlex Biosciences, respectively. (S)-[N-methyl-^3H]Reticuline and (S)-N-[N-methyl-^3H]methylcoclaurine were produced from [methyl-^3H]AdoMet (85 Ci/mmol, 3.1 TBq/mmol, Amersham Corp.) and (S)-norreticuline and (S)-coclaurine, respectively, by N-methylation of the alkaloids with S-adenosyl-L-methionine: (R,S)-tetrahydrobenzylisoquinoline N-methyltransferase (14, 15).

Construction of Mutant Berberine Bridge Enzyme Genes

The site-directed mutagenesis of the berberine bridge enzyme was performed using the pUC18 vector containing the encoding cDNA (13) into which a 3440-base pair BglII fragment from pWHA188 that contained a mutated tetracycline resistance gene (Tet^s) and the f1 origin of replication had been inserted. The oligonucleotides used for the construction of the mutant proteins were as follows: H39G, 5`-CGCAGAAAATACGGTACCATTACGAACCCC-3`; H104T, 5`-CCTTCATAACTAGTACCACCACTTCT-3`; H408S, 5`-TGTACCACTTCGCGAAGGAAACGGCGT-3`; R100T, 5`-ATGACCACCACTAGTTAATCTTATAG-3`; and Tet^R, 5`-CCACACCCGTCCTGTGGATCCTCTACGCCGGACGC-3` in which the underlined bases indicate the location of the mutation. The mutagenesis oligonucleotides were designed to introduce a restriction endonuclease recognition site so that screening for the mutants would be simplified. The DNA sequences of the mutants were verified by nucleotide sequencing(16) , and the individual berberine bridge enzyme mutated cDNAs were digested with the restriction endonucleases BglII and HindIII, introduced into the baculovirus expression plasmid pVL1393, and transfected into insect cells as described previously(13) .

Isolation of Flavinylated Peptide

Berberine bridge enzyme (1.8 nanokatal, 250 µg) was digested with endoproteinase Glu-C in 100 µl of 25 mM ammoniun acetate buffer, pH 4.0, according to the manufacturer's instructions. The digest was centrifuged for 20 min at room temperature in an Eppendorf microcentrifuge. The flavin-containing peptide was insoluble and could be collected as an intensely yellow-colored pellet after centrifugation. The pellet was redissolved in 50 µl of acetonitrile and analyzed by laser-desorption time-of-flight mass spectrometry on a Vestec BenchTop 2 spectrometer.

Berberine Bridge Enzyme Assay

The radioactive enzyme assay for the berberine bridge enzyme was performed using (S)-[N-methyl-^3H]reticuline as the substrate as described previously(2) . The conversion rates of nonradioactive substrates was determined as follows. 700 nmol of substrate were dissolved in 200 µl of 100 mM potassium phosphate buffer, pH 9.3. This solution was divided into two parts and to one part was added 0.9 nanokatal of enzyme. Both samples were incubated at 37 °C for 6 h. The samples were then lyophilized and redissolved in 1 ml of MeOH, and the products were analyzed by HPLC (column, Nucleosil C(18); 5 µm (4 times 250 mm); solvent system, A: 95% (v/v) H(2)O, 5% acetonitrile, 0.01% H(3)PO(4), B: 5% H(2)O, 95% acetonitrile, 0.01% H(3)PO(4); gradient, 0-20 min 0-100% B, 20-25 min 100% B, 25-25.1 min 100% A, 25.1-35 min 100% A; flow 1 ml/min) with detection at 280 nm.

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^2H(4) 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(4) for comparison.

Formaldehyde Trapping

The tritiated formaldehyde released upon demethylation of (S)-N-[N-methyl-^3H]methylcoclaurine (1 µCi, 85 Ci/mmol) to (S)-coclaurine by the berberine bridge enzyme (0.9 nanokatal) in a total volume of 160 µl of 20 mM potassium phosphate buffer, pH 9.3, incubated for 30 min at 37 °C was diluted by the addition of 80 µl of 37% formaldehyde and reacted with dimedone (5,5-dimethyl-1,3-cyclohexanedione) (300 mg in 3 ml of 50% methanol) at room temperature overnight. The formaldimethone crystals were collected by filtration and recrystallized to constant specific radioactivity from 50% methanol, then from 100% methanol, and finally from 100% ethanol. Control reactions were carried out without enzyme.

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 (HOO^3H and HO^3H) as well as that in the sample not treated with charcoal (tritiated formaldimethone plus HOO^3H and HO^3H) was determined by liquid scintillation counting. All values were corrected for recovery of radioactivity.


RESULTS

Coenzyme Identification and Site of Covalent Attachment

Previous characterization of the berberine bridge enzyme from Berberis species did not demonstrate a cofactor requirement, but the enzyme was shown to be sensitive to metal chelators such as EDTA(2) . Since the enzyme catalyzes a redox reaction and a cofactor would be required for catalysis, it was suggested that a metal ion might be present in the enzyme environment. With the availability of milligram quantities of recombinant enzyme, the presence Cu, Fe, Mn, Mo, and Zn ions on the enzyme were tested for by atomic absorption spectrophotometry. Although 1 mg of pure enzyme was used for each analysis, no metal was detected.

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 -2H(2)O = 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. W(3)A(4)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.

Substrate Specificity

The berberine bridge enzyme was tested for the ability to oxidize 23 isoquinoline alkaloids in an attempt to gain insight into the reaction mechanism. Of the 19 tetrahydrobenzylisoquinolines tested, the enzyme was able to catalyze formation of the berberine bridge carbon, C-8, with the following five compounds: (S)-reticuline, 13% conversion; (S)-protosinomenine, 83% conversion; (R,S)-crassifoline, 44% conversion; (R,S)-6-O-methyllaudanosoline, 10% conversion; and (R,S)-laudanosoline, 8% conversion (Table 1). In this assay, (S)-reticuline was not 100% converted because the enzymatic reaction product (S)-scoulerine inhibits the enzyme to 50% at a concentration of 10 µM. In addition, the conversion rates for (R,S)-crassifoline, (R,S)-6-O-methyllaudanosoline, and (R,S)-laudanosoline should be doubled to correct for the fact that the enzyme is specific for the S-epimer. Conversion rates of 100% for the S-epimer could be achieved for each of these substrates when more enzyme and longer incubation times were used.



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 NaB^2H(4) or NaBH(4) 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(4) (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.



Formaldehyde Trapping

With respect to the berberine bridge enzyme mechanism, the most informative enzymatic conversion was the N-demethylation of (S)-N-methylcoclaurine to (S)-coclaurine (2% conversion, Table 1). To understand how this demethylation mechanistically proceeds, the chemical nature of the leaving group was investigated. Since the berberine bridge enzyme could oxidize (S)-coreximine and (S)-norsteponine to the corresponding N-C-8 iminium ions, it was likely that (S)-N-methylcoclaurine was oxidized to the methylene iminium ion by the enzyme. This reaction product would be very unstable, and most likely the double bond would hydrate and the methyl group would then leave as formaldehyde (Fig. 4). In order to test this possibility, a formaldehyde trapping experiment was undertaken with dimedone. (S)-N-[N-methyl-^3H]Methylcoclaurine was transformed by the berberine bridge enzyme to (S)-coclaurine, and the tritiated formaldehyde thereby released was diluted with unlabeled formaldehyde and reacted with dimedone. The formaldimethone thus formed was crystallized to constant specific radioactivity: methanol-H(2)O (1:1), 839 cpm/mg; methanol, 978 cpm/mg; ethanol, 932 cpm/mg. In the absence of enzyme, the values obtained from recrystallization were: methanol: H(2)O (1:1), 30 cpm/mg; methanol, 20 cpm/mg; ethanol, 15 cpm/mg.


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-^3H]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-^3H]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(2)O(2) for each mol of (S)-reticuline converted(2) . Taken together, these results suggest that the mechanism of N-demethylation of (S)-N-[N-methyl-^3H]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.


DISCUSSION

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 ^3H 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(3) 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.




FOOTNOTES

*
This work was supported by SFB 369 of the Deutsche Forschungsgemeinschaft, Bonn, Germany. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-89-5902-242; Fax: 49-89-5902-611; ui61116{at}sunmail.lrz-muenchen.de.


ACKNOWLEDGEMENTS

We thank Dr. Christoph Eckerskorn, Martinsried, for the laser-desorption time-of-flight mass spectral measurements and Berlex Biosciences, Richmond, CA, for the kind gift pWHA188 and for the sequence of the Tet^s repair oligonucleotide. The electron spin resonance measurements were made with a Bruker ESP300 electron spin resonance spectrometer with the kind help of Dr. Torsten Linker, Würzburg.


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