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Winkler et al., Mechanism of BBE, supplementary material
1
A concerted mechanism for berberine bridge enzyme
Andreas Winkler, Andrzej Łyskowski, Sabrina Riedl, Martin Puhl,
Toni M. Kutchan, Peter Macheroux & Karl Gruber
Supplementary Material
Contents: Supplementary Figures (7 figures) 2
Supplementary Table 9
Supplementary Discussion 10
Supplementary Methods 13
Winkler et al., Mechanism of BBE, supplementary material
2
Figure S1: Schematic representation of the structure of BBE from Eschscholzia californica
(A), of glucooligosaccharide oxidase from Acremonium strictum (B, PDB entry: 1zr6), 6-
hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans (C, PDB-entry: 2bvf) and of
aclacinomycin oxidoreductase from Streptomyces galilaeus (D, PDB-entry: 2ipi). The
similarities between the four structures are evident from r.m.s-deviations of 1.5 to 1.6 Å for
330 to 370 superimposed Cα-atoms. In A, B and D the flavin cofactor is bi-covalently
attached to the protein by a histidine (to C8α) and a cysteine (to C6). In the 6-hydroxy-D-
nicotine oxidase from Arthrobacter nicotinovorans (C) the cysteine attachment is missing.
Winkler et al., Mechanism of BBE, supplementary material
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Figure S2: (a) Two perpendicular views of the Fo-Fc density map of the isoalloxazine ring
system plus His104 and Cys166 in BBE_tet contoured at 5.0 σ. (b) Active site of BBE in the
tetragonal crystal form showing His459 in two alternate conformations. (c) Two
perpendicular views of the omit density map of the isoalloxazine ring system plus His104 and
Cys166 in BBE_mon contoured at 3.0 σ. The observed density was interpreted as a
superposition of intact FAD (orange) and an intermediate product of hydrolysis in the
isoalloxazine ring system (white)1. Two partially occupied water molecules are shown as blue
spheres. (d) Time dependent degradation of the cofactor obtained during incubation in the
dark at RT in 50 mM Tris/HCl pH 9.0. Spectra were recorded after 0, 23, 65, 120 and 191 h
and are represented by the solid, dashed, dotted, dash-dotted and dash-dot-dotted line,
respectively. Spectra were corrected for protein precipitation during the long incubation time.
(e) Schematic representation of the observed degradation process under alkaline conditions.
Winkler et al., Mechanism of BBE, supplementary material
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Figure S3: (S)-Reticuline binding. (a) Stereo view of the Fo-Fc electron density map of the
bound (S)-reticuline in the active site contoured at 3.0 σ . The amino acids involved in the
covalent attachment of the cofactor were omitted for clarity. (b) Stereo view of the active site
cleft of BBE. The flavin cofactor (blue) and the substrate (S)-reticuline (yellow) are shown in
a ball-and-stick representation together with a cartoon representation and the semi-transparent
molecular surface of the protein.
Winkler et al., Mechanism of BBE, supplementary material
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Figure S4: Comparison of wild-type enzyme and the E417Q variant. Spectral changes
observed during single turnover experiments carried out with wild-type BBE (a) and the
E417Q mutant protein (b). 11 and 6 µM protein solutions, respectively, were incubated with
an excess of (S)-reticuline under anoxic conditions and spectra were recorded at various time
points. The time-course starts with the solid line and ends at the dash-dot-dotted line spanning
0.1 and 160 seconds for the wild-type and mutant protein, respectively. (c) Comparison of the
native UV-Vis absorbance spectra of wild-type (solid) and E417Q (dotted) normalized at 445
nm. (d) Kinetic traces of cofactor reduction monitored at 445 nm upon mixing ~200 µM (S)-
reticuline with ~ 9 µM protein solution (wild-type - solid, E417Q - dotted) in an anoxic
environment.
Winkler et al., Mechanism of BBE, supplementary material
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Figure S5: Spectral changes accompanying (R,S)-laudanosine oxidation to 2-methyl-3,4-
dihydropapaverinium (13). ~ 3 µM wild-type BBE was incubated with 500 µM of the
substrate analog shown in the inset. The first spectrum is represented by a solid line and
spectral changes as indicated by the arrows were recorded over a period of 2 hours (dash-dot-
dotted line).
Winkler et al., Mechanism of BBE, supplementary material
7
Figure S6: Side-product identification. HPLC analysis revealed that conversion of (S)-
reticuline (7.4 min) by E417Q results in a mixture of two products (13.8 and 14.3 min) in a
ratio of ~1:2, respectively. The major product peak represents the standard product of the
BBE catalyzed reaction [(S)-scoulerine, 14.3 min]. The additional compound produced during
this conversion was found to elute at the same time as an authentic reference for (S)-
coreximine (13.8 min). The numbers in brackets represent rounded elution times of the
substances, which are also indicated in more detail on top of each peak in the elution profiles.
The insets show the chemical structures of the two products formed.
Winkler et al., Mechanism of BBE, supplementary material
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Figure S7: Alignment of the sequences of berberine bridge enzyme from Eschscholzia
californica (Swissprot: P30986), Δ1-tetrahydrocannabinolic acid synthase (TrEMBL:
Q8GTB6) and cannabidiolic acid (CBDA) synthase (TrEMBL: A6P6V9) from Cannabis
sativa. The multiple sequence alignment was prepared using TCoffee (http://www.igs.cnrs-
mrs.fr/Tcoffee/) taking into account secondary structure elements derived from the BBE
structure. The active site residues Glu417, His459 and Tyr106 as well as the residues His104
and Cys166, which form the bi-covalent linkage of the flavin cofactor in BBE, are marked.
Winkler et al., Mechanism of BBE, supplementary material
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Table S1: Data collection and refinement statistics.
BBE_mon BBE_tet BBE_compl
Data collection
Space group C2 P41212 C2
Cell dimensions
a, b, c (Å) 99.91, 94.84, 63.98 68.52, 68.52, 246.30 98.94, 93.65, 63.16
α, β, γ (°) 90.0, 100.3, 90.0 90.0, 90.0, 90.0 90.0, 100.6, 90.0
Resolution (Å) 25.0-1.65
(1.68-1.65)* 30.0-2.05
(2.09-2.05)* 26.0-2.80
(2.85-2.80)*
Rsym 0.071 (0.278) 0.096 (0.678) 0.115 (0.424)
I / σI 26.5 (4.6) 22.9 (2.8) 7.8 (2.2)
Completeness (%) 98.7 (95.9) 96.2 (96.9) 88.9 (91.3)
Redundancy 3.7 (3.5) 6.7 (7.0) 2.9 (2.9)
Refinement
Resolution (Å) 25.0-1.65 30.0-2.05 26.0-2.8
No. reflections 69824 36664 12513
Rwork / Rfree 0.1548/0.1864 0.1845/0.2256 0.1877/0.2406
No. atoms
Protein 4098 3997 3938
Cofactor/substrate/sugars 193 106 163
Water 780 463 18
B-factors
Protein 18.7 33.1 43.3
Cofactor/substrate/sugars 24.0 44.3 57.5
Water 36.0 42.0 30.9
R.m.s. deviations
Bond lengths (Å) 0.009 0.002 0.003
Bond angles (°) 1.3 0.7 0.8
*Values in parentheses are for highest-resolution shell.
Winkler et al., Mechanism of BBE, supplementary material
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Supplementary Discussion
X-ray structure analysis
In all three structures (Table S1), 14 to 18 residues at the C terminus are missing in the
electron density. The first visible residues at the N terminus are Asp26 (in BBE_mon), Ala23
(in BBE_tet) and Ala21 (in BBE_compl). The numbering scheme is according to the
published sequence2, which includes a 23 residue signal peptide at the N terminus. Residues
in the structures with numbers smaller than 24 originate from remnants of the yeast α-factor
which was introduced for an optimal expression in Pichia pastoris3. Clear electron density
was observed for sugar moieties attached to residues Asn38 and Asn471, which were both
predicted as glycosylation sites. While only the first N-acetyl-glucosamine was visible at the
latter position, a hexasaccharide (Nag2-Man4) was placed into the electron density extending
from Asn38 in BBE_mon.
The molecular structure comprises two domains: an FAD binding domain (consisting of two
N-terminal α/β-subdomains and a C-terminal, mostly α-helical stretch) and a central α/β-
domain with a seven stranded, anti-parallel β-sheet forming the substrate binding site.
According to an analysis using the MSDssm-server4, the closest structural neighbors of BBE
are members of the p-cresol methylhydroxylase (PCMH) superfamily (Fig. S1):
glucooligosaccharide oxidase from Acremonium strictum5 (r.m.s.d.: 1.66 Å for 369 aligned
Cα-atoms), 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans6 (1.67 Å/334)
and aclacinomycin oxidoreductase from Streptomyces galilaeus7 (1.51 Å/363).
While the electron density of FAD was as expected for a nearly planar isoalloxazine ring
system in the tetragonal crystal form (Fig. S2), the density of the isoalloxazine ring in the
monoclinic structure exhibited uncommon features that were interpreted as originating from a
superposition of an intact flavin molecule and a 4a-spirohydantoin1,8 degradation product
(Fig. S2). The relatively long incubation times at pH 8.5 necessary for the growth of the
monoclinic crystals led to a partial bleaching of the intensely yellow crystallization drops and
of the crystals themselves. Control experiments under similar alkaline conditions also showed
a time dependent degradation of the flavin spectrum (Fig. S2). In the substrate complex, the
electron density around the flavin also indicates the presence of the degradation product, but
to a much smaller extent compared to the high resolution monoclinic structure. This finding is
consistent with the time elapsed between crystallization setup and data collection (five days
vs. ten weeks).
Winkler et al., Mechanism of BBE, supplementary material
11
The tetragonal crystals appeared very quickly and the diffraction dataset was collected about
two days after setting up crystallization, whereas the monoclinic crystals took weeks to grow
to usable sizes and the high resolution dataset was collected ten weeks after the setup. The
degradation of the cofactor under alkaline conditions is also interesting with respect to the
intracellular localization of BBE. Under physiological conditions BBE – with its relatively
high pH optimum9 – supposedly catalyzes its reaction in endoplasmic reticulum derived
alkaline vesicles until they fuse with the central vacuole10,11. Since no activity of BBE is
expected under the acidic conditions of the vacuole there seems to be no evolutionary
pressure to ensure cofactor stability over prolonged periods at the conditions required to
effectively catalyze its reaction. This contrasts to other members of the group of bi-covalently
flavinylated oxidases, which have an extracellular localization12,13 and therefore require a
better long term stability.
The conformations of active site residues are not significantly altered upon complexation with
(S)-reticuline. The side chain of Trp165, however, for which only weak electron density was
observed in the unbound structures, becomes better ordered in the complex and interacts with
the phenolic ring of the isoquinoline moiety.
Kinetic analysis
A detailed comparison of the biochemical properties of the E417Q to wild-type enzyme is
presented in Fig. S4 demonstrating that the UV/Vis-absorbance of the FAD cofactor is not
affected by the replacement of glutamate, which can be expected due to its relatively distant
positioning (> 6 Å). Reduction of the cofactor by (S)-reticuline is accompanied by similar
spectral changes for both the wild-type enzyme and the E417Q mutant protein, showing a
fully reduced spectrum with a broad absorption maximum around 400 nm and also the fully
oxidized spectrum of FAD is virtually identical between the two proteins.
In the case of H459A, the effects observed on kred and kcat are only up to two-fold indicating
that this amino acid does not play a major role in catalysis. On the other hand, the influence of
the Y106F replacement is more pronounced leading to a 10-fold decrease of the turnover rate
in steady-state measurements and a similar decrease of the reductive rate observed in single
turnover rapid reaction experiments of the reductive half reaction. Interestingly, the turnover
rates for the latter two muteins are affected even though their reductive rates (kred) are much
higher (H459A) or in the same range (Y106F) as the steady-state turnover (kcat) of wild-type
BBE. Since turnover is limited by the oxidative-half reaction of the cofactor in the case of the
wild-type protein14, this indicates that reoxidation might also be affected by the introduced
Winkler et al., Mechanism of BBE, supplementary material
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amino acid substitutions. Even though a detailed mechanism of the reoxidation of flavoprotein
oxidases still awaits to be elucidated, the proximity of Tyr106 and His459 to the C4a position
of the isoalloxazine ring system might point towards a role during the oxidative half
reaction15. Analysis of the cofactor reoxidation revealed that these two amino acid
substitutions indeed have an effect on the oxidative rate (Table 1, main text). In the case of
H459A, the second order rate constant for reoxidation is only half of that of the wild-type
protein, which is in line with the reduction of the turnover rate by a factor of two. The same
reduction in oxygen reactivity is observed for Y106F. In this case, however, the decrease in
the rate of cofactor reoxidation cannot explain the observed reduction for the steady-state
turnover (Table 1, main text) indicating that other processes are relevant for catalytic
efficiency.
Due to the close positioning of both Tyr106 and His459 to the newly formed carbon-carbon
bond, both amino acids could also play a role in providing the appropriate environment for
product rearomatization prior to its dissociation or the regeneration of the active site for a new
enzymatic cycle.
Enzymatic conversion of substrate analogs
The NMR-data of the reaction product obtained in a conversion of the substrate analog 8 are
as follows: 1H NMR (500 MHz, D2O): δ 8.69 (s, 1H), 7.35 (s, 1H), 7.12 (s, 1H), 4.00 (s, 3H),
4.00 (t, J = 8.2 Hz, 2H), 3.92 (s, 3H), 3.73 (s, 3H), 3.24 (t, J = 8.2 Hz, 2H); 13C NMR (125
MHz, D2O): δ 164.9, 156.6, 147.8, 132.9, 117.2, 115.0, 111.3, 56.5, 56.2, 49.5, 46.8, 24.7.
These data clearly show the generation of a double bond in conjugation to the aromatic ring
by the appearance of an additional aromatic proton signal at 8.69 ppm and by the downfield
shift of the N-methyl proton signals due to the positive charge on the nitrogen atom (4.0 ppm
compared to 2.82 ppm in case of 8).
Winkler et al., Mechanism of BBE, supplementary material
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Supplementary Methods
Chemicals and reagents
(S)-Reticuline and (S)-coreximine were from the natural product collection at the Donald
Danforth Plant Science Center. (R,S)-Laudanosine was from Extrasynthese. Oligonucleotides
were from VBC-Biotech and purified by polyacrylamide gel electrophoresis. Site-directed
mutagenesis was carried out using the QuikChange® XL kit (Stratagene). Standard chemicals
were obtained from Sigma-Aldrich.
Crystallization
Wild-type BBE was expressed in Pichia pastoris and purified as described before3. The
enzyme was crystallized at room temperature using the sitting drop vapor diffusion method
with drops of 1 μL protein solution (~30 mg mL-1 in 50 mM Tris/HCl, 150 mM NaCl, pH 9.0)
plus 1 μL reservoir solution. Diffraction quality crystals were obtained with 0.2 M MgCl2 and
30 % (w/v) PEG-4000 in 0.1 M Tris/HCl pH 8.5. Depending on the protein sample (from
different Pichia fermentations) monoclinic or tetragonal crystals appeared (Table S1). While
the latter grew overnight after microseeding, monoclinic crystals took weeks to grow to
usable sizes. For cryoprotection the crystals were transferred to a solution containing 25%
glycerol before flash-cooling in liquid nitrogen. For the soaking with substrate this solution
additionally contained 20 mM (S)-reticuline and the crystal was incubated for roughly one
minute.
Structure determination
A first complete dataset to 2.05 Å resolution was collected from a monoclinic crystal at our
in-house rotating anode generator (Cu-Kα-radiation, λ=1.5418 Å) and was used for initial
structure solution. Subsequently, datasets BBE_mon and BBE_compl were collected at
beamlines X13 (λ=0.8148 Å) and X11 (λ=0.8010 Å) at the EMBL/DESY Hamburg, whereas
dataset BBE_tet was again collected at our in-house source (Table S1). In all cases, data
reduction involved the programs DENZO and SCALEPACK16 as well as software from the
CCP4 suite17.
The structure was solved by molecular replacement with the program PHASER18 using the
first monoclinic dataset. The search model – obtained from the CaspR server19 – was a
truncated homology model of BBE mainly comprising the FAD binding domain and was
Winkler et al., Mechanism of BBE, supplementary material
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based on the structures of glucooligosaccharide oxidase from Acremonium strictum (PDB
entry: 1zr6)5, 6-hydroxy-D-nicotine oxidase from Arthrobacter nicotinovorans (PDB-entry:
2bvf)6 and aclacinomycin oxidoreductase from Streptomyces galilaeus (PDB-entry: 2ipi)7.
The overall sequence identity of these templates with BBE is about 24%. Based on this initial
phase information, the major part of the structure was automatically built using PHENIX20
and ARP/wARP21. Missing amino acids as well as the FAD cofactor could be placed in the
resulting clear difference electron density. The other structures were either solved by
molecular replacement or by rigid body refinement using the partially refined model.
The structures were further refined using the program PHENIX20. Model building and fitting
steps involved the graphics program Coot22 using σA-weighted 2Fo-Fc and Fo-Fc electron
density maps23. Rfree-values24 were computed from 5% randomly chosen reflections not used
throughout the refinement. In the higher resolution structures, water molecules were placed
automatically into difference electron density maps and were retained or rejected based on
geometric criteria as well as on their refined B-factors. In the complex structure only few well
defined water molecules were manually placed into the electron density. Details of the data
collection, processing and structure refinement are summarized in Table S1. Coordinates and
structure factors have been deposited with the Protein Data Bank under the accession numbers
3D2H (monoclinic), 3D2J (tetragonal) and 3D2D (substrate complex).
Site-directed mutagenesis
Mutagenesis was carried out with the expression vector pPICZα BBE-ER described
previously3 in order to alter amino acids potentially involved in the catalytic mechanism of
BBE. Based on the structure three amino acids in the proximity of the catalytic center were
chosen for an initial analysis. Tyr106, Glu417 and His459 were changed to Phe, Gln and Ala,
respectively, using polymerase chain reaction-based mutagenesis. The primer pairs used for
mutagenesis as described in QuikChange® XL Site-directed mutagenesis kit (Stratagene)
consisted of a sense primer (sequences indicated below) and the complementary antisense
primer. 5'-GAAGTGGTGGTCATAGTTTTGAAGGATTATCTTACACTTCTG-3' for
Y106F, 5'-CGAAGTGGTACAAGATTAATGGTTCAATATATAGTTGCCTGGAATC-3'
for E417Q and 5'-
CCAAGACTTGGGTATGTTAATGCTATTGATCTTGATCTTGGAGGGATA-3' for
H459A with the underlined codon representing the changed position. Introduction of the
expected mutation was verified by plasmid sequencing. Mutein expression and purification
followed the same procedure as for the wild-type.
Winkler et al., Mechanism of BBE, supplementary material
15
The ratio A280/A445 was virtually unaffected for both the H459A and E417Q mutant proteins
when compared with wild-type BBE indicating that the proteins are fully loaded with
cofactor. In the case of Y106F, this ratio was slightly changed due to spectral changes
indicative of 4a-spirohydantoin formation during the purification process8. It can thus be
concluded that this amino acid affects stability of the cofactor. However, since protein
concentrations were estimated based on the known extinction coefficient for the FAD cofactor
of wild-type BBE3, only the concentration of intact Y106F entered the calculations of
turnover rates.
Turnover-rate determination
For steady-state kinetic analysis the conversion of (S)-reticuline to (S)-scoulerine was
monitored by high performance liquid chromatography. Separation of the two substances was
carried out on an Atlantis® dC18 column (5 μm, 4.6×250 mm, Waters) using an isocratic
elution with 60 % MeOH/40 % 10 mM ammonium bicarbonate buffer, pH 7.0, for 11
minutes. Reaction mixtures consisted of a total volume of 200 μL with 100 µM (S)-reticuline
in 100 mM Tris/HCl pH 9.0 – the pH optimum of BBE9 – and an enzyme concentration
adjusted for each mutein in order to obtain a linear conversion rate during the initial phase of
the reaction.
Side-product identification
Identification of the additional product peak observed during turnover assays of the E417Q
mutein was performed by comparing the elution time with an authentic standard of the
suspected side-product (S)-coreximine. Slightly different conditions were used for a better
separation of the two products compared to standard activity assays. Isocratic elution with 50
% MeOH/50 % 10 mM ammonium bicarbonate buffer, pH 7.0, was carried out on an
Atlantis® dC18 column (5 µm, 4.6×150 mm, Waters) over 20 minutes.
Transient-kinetics
Determination of the reductive rates was performed as reported in a previous study14.
Apparent rate constants for the reductive half-reaction were measured at five different
concentrations of (S)-reticuline - 25, 50, 75, 125, 200 and 300 μM, and the concentration of
each mutein was ~10 μM (all concentrations are corrected for dilution after mixing in the flow
cell). The reductive rate was calculated from a hyperbolic fit to the apparent rates at all
substrate concentrations and represents the rate under saturating substrate concentrations.
Winkler et al., Mechanism of BBE, supplementary material
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Spectral changes during single-turnover experiments were monitored with a KinetaScanT
diode array detector (MG-6560) attached to the stopped-flow device (SF-61DX2 from TgK
Scientific). Rates for the oxidative half-reaction were measured by mixing substrate reduced
enzyme solution with air-saturated buffer (21 % oxygen) as described previously14.
Enzymatic conversion of substrate analogs
The substrate analog 6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline was converted
by BBE and the product identified by NMR. To that end, 2.47 mg of the latter substance were
dissolved in D2O containing 10 mM Tris/Cl and adjusted to a pD ~ 9. Conversion of the
substrate was initiated by addition of 0.5 mg recombinant BBE and monitored by TLC. After
completion of the reaction the protein was heat precipitated and after centrifugation the
supernatant was used for 1H and 13C NMR spectroscopy. The overlap of an assumed triplet
with a singlet at 4.00 ppm showing a total 5-H integral was confirmed by COSY
spectroscopy, which showed the coupling of this signal with the second triplet at 3.24 ppm.
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