Full Papers
doi.org/10.1002/cbic.202000444
ChemBioChem
free phenol as representative of a compound class very
abundant in natural environments.
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Experimental Section
Materials: All chemicals and solvents were purchased from
commercial suppliers in highest purity (p.a.). Prof. Dr. Karl-Heinz van
Pée kindly provided the plasmid pClBhis-PrnF encoding the flavin
reductase from Pseudomonas fluorescens and Prof. Dr. Werner
Hummel donated the plasmid vector pET-21_ADH encoding for
alcohol dehydrogenase. The plasmid pGro7 for the chaperone
system GroEL-GroES was obtained from TaKaRa Bio Inc. Competent
cells Escherichia coli DH5α and E. coli BL21 (DE3) were obtained
from Novagen.
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Figure 6. Specific activities of BrvH for the substrates 5-, 3-, 2-methylindole,
indole, 5-fluoro-, 5-bromo-, 5-cyano- and 5-nitroindole in comparison to the
calculated specific activities of RebH and PrnA based on the turnover
Analytical reversed-phase high performance liquid chromatogra-
phy: For analytical RP-HPLC a Shimadzu Nexera XR Luna 13 μm
À 1
À 1
[28,29]
numbers (RebH: 1.4 min , 60297 g/mol; PrnA: 1.1 min , 60000 g/mol).
C18(2) 100 Å, Column from Phenomenex was used (100×2 mm,
eluent A: H O/TFA (100:0.1), eluent B: CH CN/TFA (100:0.1), flow-
2
3
rate 650 μL/min). A linear gradient was applied starting with 95%
eluent A and 5% eluent B over 5 min to 95% eluent B and 5%
eluent A, staying for 0.5 min, then back to 95% eluent A and 5%
eluent B for 3 min.
Conclusion
Liquid chromatography–mass spectrometry: For LC-MS analysis a
Agilent Technologies 1200 with Hypersil Gold C18 (3 μm,
150x2.1 mm; eluent A: H O/CH CN/formic acid (95:5:0.1), eluent B:
Metagenomic analysis turned out to be a powerful tool for the
identification of novel flavin-dependent halogenases. A bottle-
neck of this identification technique is the lack of knowledge of
the natural substrates. In this study we tried to overcome this
issue by employing a virtual screening process, docking a
plethora of potential substrates including natural products into
the active site of BrvH generating a preliminary SAR based on
first conversion results. 137 structures were predicted for
correct BrvH active site binding. Six very promising compounds
with regard to predicted high binding energy and correct
positioning were chosen from the MOE lead-like database and
one from the IPB in-house database, of which five identical or
very similar compounds were brominated by BrvH. Interestingly,
with compounds 31 and 32, 6-membered ring aromatic
substrates different from indole were accepted. This is impor-
tant as aromatic compounds, especially phenolics, are ubi-
quitous natural products and halogenated derivatives are
common moieties in bioactive compounds. Moreover, the 3D
structure of BrvH proposed the acceptance of a bulkier
2
3
H
O/CH
CN/formic acid (5:95:0.1); flow rate: 300 μL/min; eluent A)
mass spectra Agilent
2
3
from Thermo Fisher Scientific and
a
Technologies 6220 Accurate Mass TOF/LC-MS (gas temperature:
325°C, capillary voltage: 2500 V, fragmentor voltage: 175 V) with a
Dual-ESI (spray voltage: 2.5 kV) was used. A linear gradient starting
with 100% eluent A changes over 10 min to 98% eluent B and 2%
eluent A, stays for 1 min, then over 0.5 min back to 100% eluent A
for 3.5 min.
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NMR spectroscopy: For determination of regioselectivity 1D ( H
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1
NMR, C NMR) and 2D NMR ( H, H COSY, H, H ROESY, HMQC) in
deuterated D
AV500, Bruker ( H: 500 MHz; C: 126 MHz).
-DMSO) at 25
°
C was carried out using DRX-500 and
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Vector preparation, heterologous protein expression and protein
purification: Vector expression, heterologous protein expression
and purification was carried out as described in our previous
[23]
publication.
Substrate conversion assays: Substrate assays with BrvH were
carried out with PrnF and ADH for cofactor regeneration as
[23]
substrate , and indeed even 4-n-hexylresorcinol (32) with its
lipophilic side chain can be brominated, which underlines our
hypothesis that BrvH is able to halogenate substrates with
[17]
described by Frese et al. 1.25 mg/mL enzyme was incubated with
1 mM substrate for 48 h, 500 rpm, at 25°C in 100 mM Na HPO
2
4
buffer and 1 μM FAD, 100 μM NAD, 50 mM NaBr, 2.5 U/mL PrnF,
2 U/mL RR-ADH and 5% (v/v) iso-propanol. Methanol (1:1) was
added to stop enzyme activity. Next, the samples were purified
with C18 silica (0.035–0.07 mm) for RP-HPLC and LC-MS measure-
ments.
[23]
longer alkyl chains.
The flavin-dependent halogenase BrvH accepts indole
derivatives with electron-withdrawing as well as electron-
donating groups in the six-membered ring of the indole moiety.
In a comparison of different halogenated indole substrates, it
shows the highest activity for 5-fluoroindole. Fluoride, bromide
and chloride show all a +M and À I effect, but the fluoro
substituent has the smallest van-der-Waals radius, which might
be the reason for preference of 5-fluoroindole over 5-bromo-
and 5-chloroindole.
Determination of specific activity: For determination of the specific
acitivity tests were carried out in triplicates with 50 μM substrate
and 10 μM enzyme incubated for 20 min. Every 5 min a sample was
taken, stopped with 1:1 methanol, purified on C18 silica (0.035–
0.07 mm) and analysed with RP-HPLC. With the percentage product
yield and the molecular weight of BrvH the specific activity
À 1
(
mU mg ) was calculated.
The virtual screening process not only enabled a deeper
insight into the substrate binding and SAR, but allowed the
identification of novel substrates for BrvH, including an indole-
Computational chemistry: The crystal structures of BrvH (PDB ID:
[
23]
[11]
6FRL)
and of Trp-7 halogenase PrnA (PDB ID: 2ARD)
were
and
[30]
downloaded from the protein data bank (www.rcsb.org)
subsequently prepared for the modelling studies by adding the
ChemBioChem 2020, 21, 1–8
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© 2020 The Authors. Published by Wiley-VCH GmbH
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