Biochemical evidence that berberine bridge enzyme belongs to a novel family of flavoproteins containing a bi-covalently attached FAD cofactor

Berberine bridge enzyme (BBE) is involved in the transformation of (S)-reticuline to (S)-scoulerine in benzophenanthridine alkaloid biosynthesis of plants. In this report, we describe the high level expression of BBE encoded by the gene from Eschscholzia californica (California poppy) in the methylotrophic yeast Pichia pastoris employing the secretory pathway of the host organism. Using a two-step chromatographic purification protocol, 120 mg of BBE could be obtained from 1 liter of fermentation culture. The purified protein exhibits a turnover number for substrate conversion of 8.2 s(-1). The recombinant enzyme is glycosylated and carries a covalently attached FAD cofactor. In addition to the previously known covalent attachment of the 8alpha-position of the flavin ring system to a histidine (His-104), we could also demonstrate that a covalent linkage between the 6-position and a thiol group of a cysteine residue (Cys-166) is present in BBE. The major evidence for the occurrence of a bi-covalently attached FAD cofactor is provided by N-terminal amino acid sequencing and mass spectrometric analysis of the isolated flavin-containing peptide. Furthermore, it could be shown that anaerobic photoirradiation leads to cleavage of the linkage between the 6-cysteinyl group yielding 6-mercaptoflavin and a peptide with the cysteine residue replaced by alanine due to breakage of the C-S bond. Overall, BBE is shown to exhibit typical flavoprotein oxidase properties as exemplified by the occurrence of an anionic flavin semiquinone species and formation of a flavin N(5)-sulfite adduct.     

A concerted mechanism for berberine bridge enzyme

Berberine bridge enzyme catalyzes the conversion of (S)-reticuline to (S)-scoulerine by formation of a carbon-carbon bond between the N-methyl group and the phenolic ring. We elucidated the structure of berberine bridge enzyme from Eschscholzia californica and determined the kinetic rates for three active site protein variants. Here we propose a catalytic mechanism combining base-catalyzed proton abstraction with concerted carbon-carbon coupling accompanied by hydride transfer from the N-methyl group to the N5 atom of the FAD cofactor.     

Molecular characterization of berberine bridge enzyme genes from opium poppy.

In Papaver somniferum (opium poppy) and related species, (S)-reticuline serves as a branch-point intermediate in the biosynthesis of numerous isoquinoline alkaloids. The berberine bridge enzyme (BBE) ([S]-reticuline:oxygen oxidoreductase [methylene bridge forming], EC 1.5.3.9) catalyzes the stereospecific conversion of the N-methyl moiety of (S)-reticuline into the berberine bridge carbon of (S)-scoulerine and represents the first committed step in the pathway leading to the antimicrobial alkaloid sanguinarine. Three unique genomic clones (bbe1, bbe2, and bbe3) similar to a BBE cDNA from Eschscholtzia californica (California poppy) were isolated from opium poppy. Two clones (bbe2 and bbe3) contained frame-shift mutations of which bbe2 was identified as a putative, nonexpressed pseudogene by RNA blot hybridization using a gene-specific probe and by the lack of transient expression of a chimeric gene fusion between the bbe2 5' flanking region and a beta-glucuronidase reporter gene. Similarly, bbe1 was shown to be expressed in opium poppy plants and cultured cells. Genomic DNA blot-hybridization data were consistent with a limited number of bbe homologs. RNA blot hybridization showed that bbe genes are expressed in roots and stems of mature plants and in seedlings within 3 d after germination. Rapid and transient BBE mRNA accumulation also occurred after treatment with a fungal elicitor or with methyl jasmonate. However, sanguinarine was found only in roots, seedlings, and fungal elicitor-treated cell cultures.     

Catalytic and structural role of a conserved active site histidine in berberine bridge enzyme

Berberine bridge enzyme (BBE) is a paradigm for the class of bicovalently flavinylated oxidases, which catalyzes the oxidative cyclization of (S)-reticuline to (S)-scoulerine. His174 was identified as an important active site residue because of its role in the stabilization of the reduced state of the flavin cofactor. It is also strictly conserved in the family of BBE-like oxidases. Here, we present a detailed biochemical and structural characterization of a His174Ala variant supporting its importance during catalysis and for the structural organization of the active site. Substantial changes in all kinetic parameters and a decrease in midpoint potential were observed for the BBE His174Ala variant protein. Moreover, the crystal structure of the BBE His174Ala variant showed significant structural rearrangements compared to wild-type enzyme. On the basis of our findings, we propose that His174 is part of a hydrogen bonding network that stabilizes the negative charge at the N1-C2═O locus via interaction with the hydroxyl group at C2' of the ribityl side chain of the flavin cofactor. Hence, replacement of this residue with alanine reduces the stabilizing effect for the transiently formed negative charge and results in drastically decreased kinetic parameters as well as a lower midpoint redox potential.     

Purification and characterization of the berberine bridge enzyme from berberis beaniana cell cultures

The berberine bridge-forming enzyme (BBE) has been found in 66 samples taken from differentiated plants and from cell suspension cultures. It was purified 450-fold from Berberis beaniana cell cultures by gel-filtration, DEAE and phenyl-Sepharose chromatography, electrophoresis and isoclectric focusing. The enzyme was shown to be homogeneous by gel electrophoresis (M_r = 52 kD ± 4). The enzyme, which requires the presence of oxygen, catalyses the conversion of the (S)-enantiomers of reticuline, protosinomenine and laudanosoline to the corresponding (S)-tetrahydroprotoberberines and released stoichiometric amounts of H₂O₂. Within the cells the enzyme is located in a particle with the density p = 1.14 g/ml.     

Sub-atomic resolution crystal structure of cholesterol oxidase: what atomic resolution crystallography reveals about enzyme mechanism and the role of the FAD cofactor in redox activity

The crystal structure of cholesterol oxidase, a 56kDa flavoenzyme was anisotropically refined to 0.95A resolution. The final crystallographic R-factor and R(free) value is 11.0% and 13.2%, respectively. The quality of the electron density maps has enabled modeling of alternate conformations for 83 residues in the enzyme, many of which are located in the active site. The additional observed structural features were not apparent in the previous high-resolution structure (1.5A resolution) and have enabled the identification of a narrow tunnel leading directly to the isoalloxazine portion of the FAD prosthetic group. The hydrophobic nature of this narrow tunnel suggests it is the pathway for molecular oxygen to access the isoalloxazine group for the oxidative half reaction. Resolving the alternate conformations in the active site residues provides a model for the dynamics of substrate binding and a potential oxidation triggered gating mechanism involving access to the hydrophobic tunnel. This structure reveals that the NE2 atom of the active site histidine residue, H447, critical to the redox activity of this flavin oxidase, acts as a hydrogen bond donor rather than as hydrogen acceptor. The atomic resolution structure of cholesterol oxidase has revealed the presence of hydrogen atoms, dynamic aspects of the protein and how side-chain conformations are correlated with novel structural features such as the oxygen tunnel. This new structural information has provided us with the opportunity to re-analyze the roles played by specific residues in the mechanism of the enzyme.     

Glutathione redox potential in response to differentiation and enzyme inducers

The reduced glutathione (GSH)/oxidized glutathione (GSSG) redox state is thought to function in signaling of detoxification gene expression, but also appears to be tightly regulated in cells under normal conditions. Thus it is not clear that the magnitude of change in response to physiologic stimuli is sufficient for a role in redox signaling under nontoxicologic conditions. The purpose of this study was to determine the change in 2GSH/GSSG redox during signaling of differentiation and increased detoxification enzyme activity in HT29 cells. We measured GSH, GSSG, cell volume, and cell pH, and we used the Nernst equation to determine the changes in redox potential Eh of the 2GSH/GSSG pool in response to the differentiating agent, sodium butyrate, and the detoxification enzyme inducer, benzyl isothiocyanate. Sodium butyrate caused a 60-mV oxidation (from -260 to -200 mV), an oxidation sufficient for a 100-fold change in protein dithiols:disulfide ratio. Benzyl isothiocyanate caused a 16-mV oxidation in control cells but a 40-mV oxidation (to -160 mV) in differentiated cells. Changes in GSH and mRNA for glutamate:cysteine ligase did not correlate with Eh; however, correlations were seen between Eh and glutathione S-transferase (GST) and nicotinamide adenine dinucleotide phosphate (NADPH):quinone reductase activities (N:QR). These results show that 2GSH/GSSG redox changes in response to physiologic stimuli such as differentiation and enzyme inducers are of a sufficient magnitude to control the activity of redox-sensitive proteins. This suggests that physiologic modulation of the 2GSH/GSSG redox poise could provide a fundamental parameter for the control of cell phenotype.     

Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Detection and interpretation of redox potential optima in the catalytic activity of enzymes

It is no surprise that the catalytic activity of electron-transport enzymes may be optimised at certain electrochemical potentials in ways that are analogous to observations of pH-rate optima. This property is observed clearly in experiments in which an enzyme is adsorbed on an electrode surface which can supply or receive electrons rapidly and in a highly controlled manner. In such a way, the rate of catalysis can be measured accurately as a function of the potential (driving force) that is applied. In this paper, we draw attention to a few examples in which this property has been observed in enzymes that are associated with membrane-bound respiratory chains, and we discuss its possible origins and implications for in vivo regulation.     

Role of the interface between the FMN and FAD domains in the control of redox potential and electronic transfer of NADPH-cytochrome P450 reductase

CPR (NADPH-cytochrome P450 reductase) is a multidomain protein containing two flavin-containing domains joined by a connecting domain thought to control the necessary movements of the catalytic domains during electronic cycles. We present a detailed biochemical analysis of two chimaeric CPRs composed of the association of human or yeast FMN with the alternative connecting/FAD domains. Despite the assembly of domains having a relatively large evolutionary distance between them, our data support the idea that the integrity of the catalytic cycle is conserved in our chimaeric enzymes, whereas the recognition, interactions and positioning of both catalytic domains are probably modified. The main consequences of the chimaerogenesis are a decrease in the internal electron-transfer rate between both flavins correlated with changes in the geometry of chimaeric CPRs in solution. Results of the present study highlight the role of the linker and connecting domain in the recognition at the interfaces between the catalytic domains and the impact of interdomain interactions on the redox potentials of the flavins, the internal electron-transfer efficiency and the global conformation and dynamic equilibrium of the CPRs.     

Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.     

Influence of the redox potential of the medium on the activity of polyphenol oxidase

While ascorbate is an inhibitor of polyphenol oxidase upon its activity on tyrosine, hydrogen peroxide has a promoting effect especially in conjunction with catalase. When both ascorbate and peroxide are present, the effect of the former dominates over the latter. These influences can be explained on the basis of the redox potential of the solutions: high potentials promote, while low potentials decrease the rate of the oxidative enzyme reaction. The findings might have some implications on the process of aging of tissues.     

Influence of the pH and redox potential on phosphate activity in the Parana Medio system

The effect of redox potential and pH on the phosphate mobility in two sediments were investigated using both consolidated and suspended sediments from the area where the Parana Medio long reservoir (Atgentina) is to be built (Smirnov, 1984). In addition to direct chemical sediment analysis, extraction techniques were carried out with a stepwise NH₄Cl-NaOH-HCl shaking method, the latter supposedly separating the weakly bound, the Fe- and Al- bound and the Ca- bound phosphates in the sediments.Phosphate released into water depends upon redox potential and pH, which both were modified in an experimental setup. The source of the phosphate was the fraction of Fe and/or Al bound phosphate present both in the sediment and in the suspended solids.Abbreviations cm centimeter - km kilometer - gg gram - l liter - ¬m micrometer - °C grade centigrades - km² square kilometer - m.s^–1 meter per second - m³.s^–1 cubic meter per second - mg.1¹ miligram per liter     

Analysis of promoters from tyrosine/dihydroxyphenylalanine decarboxylase and berberine bridge enzyme genes involved in benzylisoquinoline alkaloid biosynthesis in opium poppy

Tyrosine/dihydroxyphenylalanine decarboxylase (TYDC) and the berberine bridge enzyme (BBE) represent the entry point and a key branch point, respectively, in the biosynthesis of benzylisoquinoline alkaloids in select species of the Papaveraceae and Fumariaceae. Genomic clones for tydc7 and bbe1 from opium poppy (Papaver somniferum L.) were isolated. Deletion analysis of tydc7 and bbe1 5-flanking regions revealed the location of putative regulatory domains necessary for expression of the -glucuronidase (gus) reporter gene in a transient assay system based on the microprojectile bombardment of cultured opium poppy cells. A 105-nucleotide region between –393 and –287 of the tydc7 5-flanking region, and a 155-nucleotide region between –355 and –200 of the bbe1 5-flanking region, were found to be essential for promoter activity. RNA gel blot analysis showed that tydc7 and bbe1 expression is induced in cultured opium poppy cells in response to wounding or treatment with a pathogen-derived elicitor. Time-courses for the induction of tydc7 and bbe1 mRNAs in wounded cells were nearly identical to those for GUS activity in cells bombarded with select promoter-gus constructs when the –393 to –287 region of tydc7, or the –355 to –200 region of bbe1, was present. Our data suggest that the wound signal caused by the entry of DNA-coated microcarriers into opium poppy cells was sufficient to induce tydc7 and bbe1 promoter activity, and that wound-responsive regulatory elements are located within domains identified by deletion analysis.     

Influence of the redox potential on the activity of Clostridium pasteurianum and Chromatium hydrogenases

The oxidation of reduced methyl viologen by Clostridium pasteurianum or Chromatium hydrogenases as a function of the redox potential of the reaction mixture has been studied spectrophotometrically. The same results were obtained effecting the reduction of methyl viologen either with dithionite or with metallic zinc. With C. pasteurianum hydrogenase a gaussian pattern was obtained. This is indicative of a process involving two one-electron steps, which suggests that [4Fe-4S]²⁺ is the catalytically active species. On the contrary, in the case of Chromatium hydrogenase the data follow a sigmoidal pattern corresponding to a two-electron reduction process, which demonstrates that the redox site must be totally reduced to be active. This finding is at variance with the previously reported electron paramagnetic resonance spectra, which suggest that the single [4Fe-4S] cluster of this enzyme transfers or accepts only one electron.     

Functional characterization of residues involved in redox modulation of maize photosynthetic NADP-malic enzyme activity

Two highly similar plastidic NADP-malic enzymes (NADP-MEs) are found in the C(4) species maize (Zea mays); one exclusively expressed in the bundle sheath cells (BSCs) and involved in C(4) photosynthesis (ZmC(4)-NADP-ME); and the other (ZmnonC(4)-NADP-ME) with housekeeping roles. In the present work, these two NADP-MEs were analyzed regarding their redox-dependent activity modulation. The results clearly show that ZmC(4)-NADP-ME is the only one modulated by redox status, and that its oxidation produces a conformational change limiting the catalytic process, although inducing higher affinity binding of the substrates. The reversal of ZmC(4)-NADP-ME oxidation by chemical reductants suggests the presence of thiol groups able to form disulfide bonds. In order to identify the cysteine residues involved in the activity modulation, site-directed mutagenesis and MALDI-TOF (matrix-assisted laser desorption ionization-time of flight) analysis of ZmC(4)-NADP-ME were performed. The results obtained allowed the identification of Cys192, Cys246 (not conserved in ZmnonC(4)-NADP-ME), Cys270 and Cys410 as directly or indirectly implicated in ZmC(4)-NADP-ME redox modulation. These residues may be involved in forming disulfide bridge(s) or in the modulation of the oxidation of critical residues. Overall, the results indicate that, besides having acquired a high level of expression and localization in BSCs, ZmC(4)-NADP-ME displays a particular redox modulation, which may be required to accomplish the C(4) photosynthetic metabolism. Therefore, the present work could provide new insights into the regulatory mechanisms potentially involved in the recruitment of genes for the C(4) pathway during evolution.     

An intact eight-membered water chain in drosophilid alcohol dehydrogenases is essential for optimal enzyme activity

All drosophilid alcohol dehydrogenases contain an eight-member water chain connecting the active site with the solvent at the dimer interface. A similar water chain has also been shown to exist in other short-chain dehydrogenase/reductase (SDR) enzymes, including therapeutically important SDRs. The role of this water chain in the enzymatic reaction is unknown, but it has been proposed to be involved in a proton relay system. In the present study, a connecting link in the water chain was removed by mutating Thr114 to Val114 in Scaptodrosophila lebanonensis alcohol dehydrogenase (SlADH). This threonine is conserved in all drosophilid alcohol dehydrogenases but not in other SDRs. X-ray crystallography of the SlADH(T114V) mutant revealed a broken water chain, the overall 3D structure of the binary enzyme-NAD(+) complex was almost identical to the wild-type enzyme (SlADH(wt) ). As for the SlADH(wt) , steady-state kinetic studies revealed that catalysis by the SlADH(T114V) mutant was consistent with a compulsory ordered reaction mechanism where the co-enzyme binds to the free enzyme. The mutation caused a reduction of the k(on) velocity for NAD(+) and its binding strength to the enzyme, as well as the rate of hydride transfer (k) in the ternary enzyme-NAD(+) -alcohol complex. Furthermore, it increased the pK(a) value of the group in the binary enzyme-NAD(+) complex that regulates the k(on) velocity of alcohol and alcohol-competitive inhibitors. Overall, the results indicate that an intact water chain is essential for optimal enzyme activity and participates in a proton relay system during catalysis.     

NADPH-cytochrome P-450 reductase. Physical properties and redox behavior in the absence of the FAD moiety

NADPH-cytochrome P-450 reductase contains one molecule each of FMN and FAD. The FAD moiety has been selectively removed, producing the FMN reductase. The FMN reductase is stable and enzymatic activity is reconstituted with either FAD or FMN. FMN remains tightly bound, but can both dissociate from the FMN site and bind to the vacant FAD site. The amount of FMN bound in the FAD site is minimal under specific experimental conditions. There are at least two conformational subpopulations of the FMN reductase; NADP dissociates readily from one but extremely slowly from the other. Rapid dissociation of NADP is regained upon reconstitution with FAD. The one-electron redox state of the FMN reductase is thermodynamically stabilized, though to a lesser degree than in the holoreductase. When two-electron reduced FMN reductase is exposed to oxygen, a stable species with an absorbance peak at 580 nm forms rapidly and quantitatively. This species has been identified by electron paramagnetic resonance spectroscopy as the neutral radical of FMN and is indistinguishable from the air-stable radical of the holoreductase. The redox behavior of the FMN reductase is in agreement with properties proposed previously for the FMN site.     

The presence of a hydrogen bond between asparagine 485 and the pi system of FAD modulates the redox potential in the reaction catalyzed by cholesterol oxidase.

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. An asparagine residue (Asn485) at the active site is believed to play an important role in catalysis. To test the precise role of Asn485, we mutated it to a leucine and carried out kinetic and crystallographic studies. Steady-state kinetic analysis revealed a 1300-fold decrease in the oxidation k(cat)/K(m) for the mutant enzyme whereas the k(cat)/K(m) for isomerization is only 60-fold slower. The primary kinetic isotope effect in the mutant-catalyzed reaction indicates that 3alpha-H transfer remains the rate-determining step. Measurement of the reduction potentials for the wild-type and N485L enzymes reveals a 76 mV decrease in the reduction potential of the FAD for the mutant enzyme relative to wild type. The crystal structure of the mutant, determined to 1.5 A resolution, reveals a repositioning of the side chain of Met122 near Leu485 to form a hydrophobic pocket. Furthermore, the movement of Met122 facilitates the binding of an additional water molecule, possibly mimicking the position of the equatorial hydroxyl group of the steroid substrate. The wild-type enzyme shows a novel N-H...pi interaction between the side chain of Asn485 and the pyrimidine ring of the cofactor. The loss of this interaction in the N485L mutant destabilizes the reduced flavin and accounts for the decreased reduction potential and rate of oxidation. Thus, the observed structural rearrangement of residues at the active site, as well as the kinetic data and thermodynamic data for the mutant, suggests that Asn485 is important for creating an electrostatic potential around the FAD cofactor enhancing the oxidation reaction.