Kinetic characterisation of the FAD dependent monooxygenase TropB and investigation of its biotransformation potential

Article abstract of DOI:10.1039/c5ra06693j

Achieving regio-specific hydroxylation of aromatic compounds remains a major challenge in synthetic chemistry. By contrast, this transformation is readily accomplished in nature through the action of FAD-dependant monooxygenase enzymes. Here, we report th

Full text of DOI:10.1039/c5ra06693j

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Kinetic characterisation of the FAD dependent
monooxygenase TropB and investigation of its
biotransformation potential†
Cite this: RSC Adv., 2015, 5, 49987
Amira Abood,‡ab Ahmed Al-Fahad,‡ac Alan Scott,a Alaa El-Dein M. S. Hosny,d
Amal M. Hashem,b Azza M. A. Fattah,b Paul R. Race,e Thomas J. Simpsona
af
*
and Russell J. Cox
Achieving regio-specific hydroxylation of aromatic compounds remains a major challenge in synthetic
chemistry. By contrast, this transformation is readily accomplished in nature through the action of FAD-
dependant monooxygenase enzymes. Here, we report the kinetic characterisation of one such enzyme,
TropB, from the stipitatic acid biosynthetic pathway. Analogues of the TropB natural substrate, 3-methyl-
orcinaldehyde, were synthesised and used to examine the substrate selectivity of this enzyme. TropB
displays broad substrate tolerance, for instance accepting single-ring aromatic substrates containing a
range of C-1 substituents with varying electronic and steric properties. These include nitro, nitrosyl, alkyl,
and aryl keto groups. Bicyclic substrates, however, were rejected by TropB. Additionally, C-5 substituents
on single-ring aromatic substrates were not tolerated whereas the presence of a 6-methyl group was
found to be important for substrate binding. Docking studies were employed to investigate and
understand the broad substrate selectivity of TropB and identifies the key structural elements of its
substrates. Our work has shown that TropB is an attractive target for biocatalyst engineering and
industrial aromatic hydroxylation.
Received 14th April 2015
Accepted 27th May 2015
DOI: 10.1039/c5ra06693j
www.rsc.org/advances
large-scale production of 3-tert-butylcatechol,3 a precursor for
many important chemicals and synthetically intractable targets.
Introduction
The recent elucidation of the biosynthetic pathway for stip-
itatic acid led to the identication of a new FMO (TropB).4 This
enzyme catalyses the oxidative dearomatisation of 3-methyl-
orcinaldehyde 1 to produce a dienone 2 (Scheme 1A). This
hydroxylation step is remarkably similar to one of the steps
reported for the chemical synthesis of azaphilones and their
analogues (Scheme 1B).5,6
The synthetic step requires extreme cold temperatures (ꢀ70
to ꢀ10 ꢁC), hazardous reagents and relatively long reaction time
(30 h). It would be advantageous if related transformations
could be performed bio-catalytically using TropB or a related
enzyme. This would obviate the utilisation of dangerous
chemicals and challenging reaction conditions to facilitate the
synthesis of key synthetic intermediates.
The direct hydroxylation of aromatic compounds in a chemo-,
regio- and stereo-selective manner remains a major challenge in
synthetic organic chemistry and is “one of the most challenging
elds in modern synthesis”.1 In contrast, such reactions are
rapid and efficient in nature. Metabolic pathways oen involve
aromatic hydroxylases and such enzymes could be potentially
engineered and exploited for chemo-synthetic processes. In
particular avin dependant mono-oxygenases (FMOs) are
attractive candidates for industrial aromatic hydroxylation.2 For
example, an FMO from Pseudomonas azelaica (2-hydroxy-
biphenyl 3-mono-oxygenase) was articially re-engineered for a
aSchool, of Chemistry, University of Bristol, Cantock's Close, BS8 1TS, UK. E-mail:
russell.cox@oci.uni-hannover.de
In our previous work, TropB was overproduced recombi-
nantly in E. coli and puried by nickel affinity chromatography,
eluted as a single SDS-PAGE band (see ESI†). The puried
protein was shown by UV-Vis spectrophotometry and LC-MS to
possess a non-covalently bound FAD cofactor. Exogenous NADH
or NADPH is required as an electron donor for in vitro activity.
Kinetic analysis showed that the enzyme possesses a slight
preference for NADPH.4 The enzyme was shown to hydroxylate 1
regio-selectively at C-3 to give the 3R congured product.
Different substrates including 3-methylorsellinic acid 3 and a
bChemistry of Natural and Microbial Products Department, National Research Centre,
Cairo, 12622, Egypt
cChemistry Department, Faculty of Science, Al Baha University, 1988, Al Baha, Saudi
Arabia
dMicrobiology Department, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt
eSchool of Biochemistry, University of Bristol, Medical Sciences Building, BS8 1TD, UK
f
¨
¨
Institut fur Organische Chemie, Leibniz Universitat Hannover, Schneiderberg 1B,
30167, Hannover, Germany
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra06693j
‡ These authors contributed equally.
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activity. The identity of the puried protein was conrmed by
MALDI-TOF MS of a tryptic digest (see ESI†).
The effect of temperature and pH on TropB activity was
investigated using the enzyme's natural substrate, 3-methyl-
orcinaldehyde 1. This was to determine the optimum reaction
conditions for later use during more detailed kinetic analyses.
The enzyme was incubated with a xed concentration of
substrate (200 mM) and NADPH (100 mM) at different tempera-
tures and appropriately buffered pHs and the initial rate of
reaction measured.
TropB activity was monitored spectrophotometrically by
following the formation of the reaction product at 400 nm. The
pH at which TropB was optimally active was pH 8.0. There was a
considerable decrease in enzyme activity below pH 7 or above
pH 9 (Fig. 2A). The optimum temperature for Trop B activity was
ꢁ
found to be 30 C although surprisingly the enzyme did retain
Scheme 1 (A) TropB catalysed reaction; (B) synthetic step reported for
the synthesis of azaphilones.
ꢁ
40% of maximum activity at 60 C (Fig. 2B).
Synthesis of substrate analogues
Compounds 15–27 (Fig. 3), were designed to probe the substrate
selectivity of TropB in addition to the previously tested
substrates. Metabolites 25–27 are natural products and were
extracted from their natural sources according to established
protocols.5,7 The analogues 4 and 8 were purchased from Sigma.
The remaining compounds were produced synthetically.
Aryl alkyl ketones were obtained directly from Friedel–Cras
acylation using 2,5-dimethylresorcinol 9 with the appropriate
acid chlorides in the presence of zinc powder as a catalyst
(Scheme 2).8 The yield of desired products was very low (<5%).
This was unsurprising given the presence of unprotected
Fig. 1 Different compounds incubated with TropB.
number of analogous compounds 4–14 (Fig. 1) were tested with
TropB. Only the methyl ketone 4 was hydroxylated, whereas the
other substrates were not. Here we extend these initial investi-
gations by performing a detailed kinetic characterisation of
TropB, to explore the enzyme's tolerance to a broad range of
alternative aromatic substrates for example, compounds that
differ in alkyl chain length and branching, and different
substituents on the aromatic ring. Additionally, the effects of
temperature and pH on the enzyme activity were also investi-
gated in order to identify the optimum reaction conditions for
TropB.
Results and discussion
Overproduction of TropB and assay optimisation
N-terminally hexahistidine tagged TropB was produced by
recombinant overexpression in E. coli BL21-CodonPlus (DE3)-
RP and puried to homogeneity using a combination of Ni-
affinity and size exclusion chromatography (ESI Fig. S1†). The
puried protein had a distinctive yellow colour and exhibited
typical avoprotein absorbance spectra with maxima at 368 and
455 nm. These observations were consistent with those reported
Fig. 2 The relative effect of (A) pH and (B) temperature on TropB
previously.4 No exogenous avin was required to detect enzyme activity with 3-methylorcinaldehyde 1.
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Fig. 3 Structures of the compounds for evaluating the substrate selectivity of TropB.
hydroxyl groups within 9 leading to the formation of many side LCMS analysis was used to detect the presence of reaction
products which were detected by LC-DAD-MS. However, the products. Control experiments with heat-denatured protein
desired products were easily identied by their mass and UV were also performed. Compounds which were demonstrated as
absorption which was higher than those of the oxygen-acylated substrates by TropB in this assay were then taken forward for
side products. As the aim was to rapidly produce only a few full kinetic characterisation. All kinetic assays were performed
milligrams of material for enzyme assays the low yields were not using a continuous spectrophotometric assay monitoring either
problematic. The only exception was for 15 where it was not product formation at 400 nm or conversion of NADPH to NADP+
possible to generate enough material for full kinetic analysis.
at 340 nm (Table 1) to determine the initial rate.
The syntheses of 4-nitro-2,5-dimethylresorcinol 22 and 4,6-
TropB was found to catalyse the NADPH dependent conver-
dinitro-2,5-dimethylresorcinol 24 were achieved by the mono sion of the natural substrate 3-methylorcinaldehyde 1 to the
and di-nitration of 9 using a mixture of diluted nitric acid and dienone 2 with a kcat of 0.298 ꢂ 0.006 sꢀ1. This compares
acetic anhydride.9,10 The 4-nitroso-2,5-dimethylresorcinol unfavourably to other FMO enzymes such as 2-methyl-3-
compound 23 was synthesised using diluted sulphuric acid hydroxypyridine-5-carboxylic-acid dioxygenase13,14 (MHPCO)
and sodium nitrite following a previously reported synthesis.11 which has a kcat of 11.8 sꢀ1 and 3-hydroxybenzoate 6-hydroxy-
All the compounds were puried using preparative LC-MS lase14 (3HB4H) which has a kcat of 36 sꢀ1. However, the KM
and the structure of each was conrmed by high resolution determined for TropB (20.0 ꢂ 1.42 mM) is similar to the values
mass spectrometry and NMR. To the best of our knowledge published for MHPCO (25.4 mM) and 3HB4H (27 mM).
none of these compounds have previously been characterised
The acetophenone derivative 4 is turned over by the enzyme
and only 23 and 15 had previously been synthesised.10,12 Char- at a similar rate to the natural substrate but binds more weakly
acterisation of compounds is given in the ESI.†
as indicated by a 9-fold increase in KM. This is probably due to
the missing 6-methyl group because compounds 16 and 17
which retain the 6-methyl group have lower KM values than 4.
The fact that these KM values are still higher than the natural
substrate is probably due to steric hindrance from the longer
carbon chains. This less than optimal binding was probably the
reason for the lower kcat values. Compound 8 which lacks both
methyl groups was not accepted by the enzyme.
Substrate selectivity
TropB and its potential substrates (2.5 mM) were incubated ꢁfor
1 hour with an excess of NADPH (3.0 mM) at pH 8 and 30 C.
Compounds 18–21 which have long or branched alkyl ketone
chains and aryls on the C-1 position are not well tolerated by the
enzyme and yielded un- or barely-detectable levels of reaction
product. The nitro and nitroso derivatives 22 and 23 were
accepted as substrates but the turnover was slower than for the
natural substrate and was similar to compounds 16 and 17. The
KM value for these compounds was higher than the natural
Scheme 2 Friedel–Crafts acylation using acyl chlorides with aromatic
compounds and Zn powder.
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Table 1 Substrate tolerance and kinetic parameters of TropB with the substrates tested. All kinetic assays were done in triplicate. “Trace” means
the amount of product detected was at the lower limit of detection. Errors show standard deviation. n.d. not determined
Turnover detected
on LCMS aer 1 h
Substrate
kcat/sꢀ1
KM/mM
kcat/KM/mMꢀ1 sꢀ1
1
3
4
5–14
15
100%
No activity
100%
No activity
n.d.
0.298 ꢂ 0.006
20.0 ꢂ 1.42
15.0 ꢂ 0.97
—
—
—
0.178 ꢂ 0.006
188.8 ꢂ 31.34
0.958 ꢂ 0.1
—
—
—
—
—
—
16
17
18
100%
100%
16%
0.040 ꢂ 0.002
79.7 ꢂ 9.17
90.2 ꢂ 22.3
—
0.46 ꢂ 0.03
0.85 ꢂ 0.16
—
0.074 ꢂ 0.005
—
19–21
22
23
Trace
100%
100%
No activity
—
—
—
0.020 ꢂ 0.003
0.013 ꢂ 0.0005
—
79.0 ꢂ 30.2
158.8 ꢂ 24.6
—
0.255 ꢂ 0.05
0.08 ꢂ 0.01
—
24–27
substrate and nitroso had a higher KM than the nitro analogue.
To determine the substrate binding mode and possible
26 and 27 are substrates for SorbC, a homologue of TropB, and catalytic mechanism, a sequence alignment was made between
these were chosen for analysis along with 24 and 25.7 These TropB and HB6H (Fig. 5). Two residues involved in substrate
compound were not substrates for TropB.
interactions in 4bjy are conserved in TropB and could play
Attempts to further purify and characterise the oxidation similar roles. These are Y217 (¼ Y239) which H-bonds to the C1
products failed, since they were not stable in organic solvents.15 substituent and H213 (¼ H235) which was proposed to act as a
However oxidation of the nitroso compound 23 resulted in the base to deprotonate the phenolic hydroxyl. The latter was shown
formation of the stable oxime 28 (Fig. 4). The reaction using 23 to be essential for catalysis in HB6H by mutagenesis studies.
was scaled up to 16 ml and the product 28 puried by LC-MS.
The TropB substrate 1 was manually docked into the
Seven milligrams of the product (99%) was obtained as a col- homology model near the FAD in an orientation consistent with
ourless oil. The molecular formula C8H9NO4 was deduced from the known stereo-chemical outcome of the reaction.4 The
the HRMS data (found: 206.0425 [M]Na+; calcd: 206.0429 for resulting protein-ligand complex was energy minimised using
C8H9NNaO4). Analysis of the 1H and 13C-NMR spectra of the the YASARA algorithm (Fig. 6). P329 and I237 make hydro-
product demonstrated that oxidation occurred via hydroxyl- phobic interactions with the substrate and F252 directly inter-
ation at C-2 of the benzenoid ring (see ESI† for data).
acts with substrate 6-methyl group. N248 is also within H-
˚
In an attempt to rationalise, the substrate selectivity of TropB bonding distance of the carbonyl oxygen (2.4 A). The substrate
a homology model of the enzyme was generated. TropB shows and FAD are orientated approximately perpendicular to each
25% identity to the FAD-dependent 3-hydroxybenzoate 6- other which is similar to the arrangement of these moieties in
hydroxylase (HB6H) from Rhodococcus jostii RHA1 for which a HB6H.17
crystal structure has been obtained (pdb accession 4bjy).16 This
˚
Y239 is located near the 2-hydroxy of the substrate (2.4 A)
˚
was used as a template for the development of a threaded model and the carbonyl oxygen (1.9 A), while H235 is near the
˚
structure using the SWISS-MODEL protein structure homology- substrate 4-hydroxy (within 4.5 A). These two residues could
modelling server. The FAD cofactor was placed into the active therefore play a role in catalysis (Scheme 3). Deprotonation of
site of the homology model by transferring the coordinates from the 4-hydroxy by H235 would increase the nucleophilicity of the
the template (4bjy) into the homology model and subjecting the 3-carbon thus aiding in its attack of the FAD hydroperoxide. The
protein-ligand complex to energy minimisation.
tyrosine could then act as a general acid protonating the
resulting alkoxide during or aer reaction. Such an acid-base
mechanism would be consistent with the pH prole (Fig. 2)
which shows an optimum at pH 8.0.
This mechanism would also be consistent with TropB
accepting nitro and nitroso derivatives and would provide an
explanation for why TropB rejects the carboxylate derivative 3 as
Fig. 4 Conversion of 23 to 28 by TropB over 40 minutes showing Fig. 5 Alignment of TropB and HB6H (4bjy) showing the conserved
consumption of 23 and formation of 28.
His and Tyr residues involved in substrate interactions.
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Fig. 6 Docking of 3-methylorcinaldehyde inside the active site of the
TropB homology model. His-235 and Tyr-239 are conserved from the
template (4bjy). His235 to H (hydroxyl) ¼ either 4.5 A or 6.2 A˚
depending on the orientation of the H. After energy minimisation the H
is 6.2 A˚ away from the His but if the H is rotated to point towards the
His then this is reduced to 4.5 A˚. Colour scheme: yellow, cofactor;
green potential catalytic residues; grey; hydrophobic and H-bonding
interactions.
Fig.
7
Active site surface of TropB homology model coloured
according to electrostatic potential. (A) shows the boundary of the
active site surface when the natural substrate (green) was docked. This
active site could accommodate up to 3 carbons beyond the carbonyl
oxygen. (B) shows the extra space opened up by conformational
change during energy minimisation when 19 (grey) was docked.
this is clearly not sufficient to allow 19 or the aromatic
substrates 20 and 21 to be efficient substrates. The active site
surface also appears to be highly charged and thus is not
favourable for binding hydrophobic groups. The kinetic data
showed a role for the 6-methyl group in binding and the model
shows this group interacts with F252, possibly xing the
substrate in the correct orientation for effective deprotonation
by H235.
Conclusions
In summary, we have identied the optimum reaction condi-
tions for the FAD dependent monooxygenase TropB and probed
the substrate selectivity of this enzyme using a range of newly
synthesised substrate analogues. We have identied the key role
of a 6-methyl group for substrate binding within the TropB
active site. Nitro and nitroso containing substrates are tolerated
by the enzyme as are aryl alkyl ketones with chains of 4 carbons
or less. Substrates containing branched chains and bicyclic
systems are not tolerated. These insights will be of value in
guiding the design of new TropB substrates for enzymatic
conversion to high-value chemicals and future efforts to engi-
neer the substrate selectivity of these enzymes, thus illustrating
the potential utility of TropB as a potential biocatalyst.
Scheme 3 Possible mechanism for the hydroxylation of 1 catalysed by
TropB.
the carboxylate ion would make deprotonation of the phenolic
hydroxyl groups by H235 more difficult.
Experimental
Chemicals
Consistent with our kinetic analyses, the active site in our
model is large enough to accommodate alkyl chains of up to 3
carbons beyond the carbonyl oxygen (Fig. 7), but not the bulkier
aromatic substrates. Interestingly, docking of bromoalkyl
substrate 19 shows that the active site pocket could reorganise
to increase the available volume for some substrates, although
FAD, NADPH, 2,4-dihydroxy-3,5-dimethylacetophenone and 2,4-
dihydroxy benzaldehyde were purchased from Sigma. NMR was
performed using a Varian VNMRS 400 or Varian VNMRS 500
spectrometer. High resolution mass spectrometry was achieved
with a Bruker Daltonics Apex IV FT-ICR.
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Bioinformatic analysis
ꢂ1-(2-Methylbutanoyl)-2,4-dihydroxy-3,6-dimethylbenzene 18.
White needles, 3% yield; m.p. 90 ꢁC; IR (neat): nmax 3367, 1598,
1457, 1415, 1227 and 1117 cmꢀ1; UV (CH3CN/H2O) lmax 223 and
291 nm; 1H-NMR (500 MHz, CDCl3) dH ¼ 12.74 (br, 1H, 2-OH),
6.21 (s, 1H, 5-H), 2.88 (m, 1H, 10-H), 2.53 (3H, s, 8-H), 2.09 (3H,
Similar amino acid sequences were identied by a BLASTP
search (http://blast.ncbi.nlm.nih.gov). Multiple sequence
alignments of TropB were performed using Multalin (http://
multalin.toulouse.inra.fr/multalin/)17 and formatted by
ESPript (http://espript.ibcp.fr/ESPript/ESPript/).18
3
s, 7-H), 1.79 (m, 1H, 11-Hb), 1.46 (m, 1H, 11-Ha), 1.17 (d, J ¼
3
6.7, 3H, 13-H), 0.88 (t, J ¼ 7.4, 3H, 12-H); 13C-NMR (125 MHz,
CDCl3) dC ¼ 212.6 (C-9), 163.1 (C-2), 158.0 (C-4), 137.6 (C-6),
116.1 (C-1), 111.0 (C-5), 109.0 (C-3), 46.0 (C-10), 27.9 (C-11),
24.0 (C-8), 17.1 (C-13), 12.0 (C-12), 7.7 (C-7); HRMS for
Isolation of natural products
3-Methylorcinaldehyde 1 and 5-hydroxy-3-methylorcin-aldehyde
25 were isolated from T. stipitatus knockout strains DtsL1 and
the dienone 2 was isolated from DtsR5 by LC-MS as described
before.8 Dihydrosorbicillin 26 and 2,3-sorbicillin 27 were iso-
lated from Penicillium chrysogenum as described before.7
C
13H19O3 calcd: 223.1334; found: 223.1330 [M]H+.
1-(6-Bromohexanoyl)-2,4-dihydroxy-3,6-dimethylbenzene
19.
ꢁ
White needles, 4% yield; m.p. 94–96 C; IR (neat): nmax 3376,
1729, 1601, 1415, 1117 and 746 cmꢀ1; UV (CH3CN/H2O) lmax
222 and 289 nm; 1H-NMR (400 MHz, CDCl3) dH ¼ 13.51
3
(br, 1H, 2-OH), 6.21 (s, 1H, 5-H), 3.43 (t, J ¼ 6.6, 2H, 14-H),
2.92 (t, 3J ¼ 7.3, 2H, 10-H), 2.53 (3H, s, 8-H), 2.09 (3H, s, 7-H),
1.90 (m, 2H, 13-H), 1.76 (m, 2H, 11-H), 1.51 (m, 2H, 12-H);
13C-NMR (100 MHz, CDCl3) dC ¼ 206.8 (C-9), 164.3 (C-2), 158.3
(C-4), 138.5 (C-6), 115.5 (C-1), 111.2 (C-5), 109.1 (C-3), 43.9
(C-10), 33.8 (C-14), 32.8 (C-13), 28.0 (C-12), 25.1 (C-8), 24.2
(C-11), 7.7 (C-7); HRMS for C14H20BrO3 calcd: 315.0596; found:
315.0602 [M]H+.
Chemical synthesis
Aryl alkyl ketones. Aryl alkyl ketones were synthesised by
Friedel–Cras acylation using acyl chlorides and aromatic
compounds according to established protocol.9 The reaction
was performed on a small scale with 13.8 mg (0.1 mmol) of 2,5-
dimethyl-resorcinol and 6.5 mg (0.1 mmol) of very ne zinc
powder in a 5 ml reaction vial. Particles were ground together
using a small glass stirring rod before the addition of 0.11 mmol
of an alkyl or aryl acyl chloride. This was mixed for 5 minutes at
room temperature. The reaction was quenched with 2 ml of
methanol. The solution was ltered and subjected directly to
LC-DAD-MS for analysis and purication.
1-Benzoyl-2,4-dihydroxy-3,6-dimethylbenzene 20. Colourless
needles, 3% yield; m.p. 145 ꢁC; UV (CH3CN/H2O) lmax 203, 248
1
and 304 nm; H-NMR (400 MHz, CDCl3) dH ¼ 12.74 (br, 1H, 2-
OH), 7.59 (dd, 3J ¼ 8.4, 5J ¼ 1.2, 2H, 11-H and 15-H), 7.53 (tt, 3J ¼
7.4, 4J ¼ 1.4, 1H, 13H), 7.44 (m, 2H, 12-H and 14-H), 6.20 (s, 1H,
5-H), 2.16 (3H, s, 7-H), 1.84 (3H, s, 8-H); 13C-NMR (100 MHz,
CDCl3) dC ¼ 201.8 (C-9), 161.6 (C-2), 158.5 (C-4), 141.6 (C-10),
138.9 (C-6), 132.1 (C-13) 128.7 (C-11 and C-15), 128.6 (C-12 and
C-14), 115.7 (C-1), 110.5 (C-5), 108.8 (C-3), 23.4 (C-8), 7.7 (C-7);
HRMS for C15H15O3 calcd: 243.1021; found: 243.1032 [M]H+.
1-(2,4-Dimethoxybenzoyl)-2,4-dihydroxy-3,6-dimethylbenzene 21.
Colourless needles, 2% yield; m.p. 155 ꢁC; UV (CH3CN/H2O) lmax
204 and 304 nm; 1H-NMR (500 MHz, CD3CN) d ¼ 12.65 (br, 1H, 2-
OH), 7.16 (d, 3J ¼ 8.7, 1H, 15-H), 6.59 (dd,3J ¼ 8.7, 4J ¼ 2.5, 1H, 14-
H), 6.58 (s, 1H, 12-H), 6.19 (s, 1H, 5-H), 3.84 (s, 3H, 17-H), 3.72 (s,
3H, 16-H), 1.80 (3H, s, 8-H), 1.47 (3H, s, 7-H); 13C-NMR (125 MHz,
CD3CN) d ¼ 201.0 (C-9), 164.1 (C-2), 164.0 (C-13), 161.1 (C-4),
158.9 (C-11), 140.9 (C-6), 130.7 (C-15), 126.0 (C-10), 116.5 (C-1),
111.4 (C-5), 109.6 (C-3), 106.3 (C-14), 99.6 (C-12), 56.5 (C-17),
56.3 (C-16), 22.5 (C-8), 7.9 (C-7); HRMS for C15H15O3 calcd:
303.1232; found: 303.1239 [M]H+.
1-Acetyl-2,4-dihydroxy-3,6-dimethylbenzene 15 (ref. 19). White
needles; 5% yield; m.p. 152 C (lit.20 149–150 C); UV (CH3CN/
ꢁ
ꢁ
H2O), lmax 222 and 290 nm; IR (neat): nmax 3397, 1598, 1457,
1415, 1227 and 1117 cmꢀ1; H-NMR (400 MHz, CD3CN) dH
¼
1
13.82 (br, 1H, 2-OH), 6.27 (s, 1H, 5-H), 2.59 (s, 3H, 10-H), 2.52 (s,
3H, 8-H), 1.98 (3H, s, 7-H); 13C-NMR (100 MHz, CD3CN) dC
206.0 (C-9), 165.5 (C-2), 160.9 (C-4), 140.8 (C-6), 115.5 (C-1),
111.8 (C-5), 109.8 (C-3), 33.4 (C-10), 25.0 (C-8), 7.9 (C-7); HRMS
for C10H13O3 calcd: 181.0865; found: 181.0860 [M]H+.
¼
1-Propionyl-2,4-dihydroxy-3,6-dimethylbenzene 16. White nee-
dles, 4% yield; m.p. 88 ꢁC; IR (neat): nmax 3397, 1601, 1420, 1236,
1108 and 1075 cmꢀ1; UV (CH3CN/H2O) lmax 222 and 286 nm; 1H-
NMR (400 MHz, CD3CN) dH ¼ 13.50 (br, 1H, 2-OH), 6.29 (s, 1H,
5-H), 2.96 (m, 2H, 10-H), 2.51 (3H, s, 8-H), 1.98 (3H, s, 7-H), 1.13
(t, 3J ¼ 7.07, 3H, 11-H); 13C-NMR (100 MHz, CD3CN) dC ¼ 209.0
(C-9), 164.8 (C-2), 160.4 (C-4), 140.0 (C-6), 115.6 (C-1), 111.8 (C-
5), 109.8 (C-3), 37.9 (C-10), 25.1 (C-8), 9.1 (C-11), 7.9 (C-7);
HRMS for C11H15O3 calcd: 195.1021; found: 195.1026 [M]H+.
1-Butanoyl-2,4-dihydroxy-3,6-dimethylbenzene 17. White nee-
dles, 5% yield; m.p. 110 ꢁC; IR (neat): nmax 3457, 1598, 1403,
1300, 1207 and 1111 cmꢀ1; UV (CH3CN/H2O) lmax 223 and 288
nm; 1H-NMR (400 MHz, CDCl3) dH ¼ 13.54 (br, 1H, 2-OH), 6.20
(s, 1H, 5-H), 2.88 (t, 3J ¼ 7.30, 2H, 10-H), 2.54 (3H, s, 8-H), 2.09
(3H, s, 7-H), 1.76 (m, 2H, 11-H), 0.98 (t, 3J ¼ 7.40, 3H, 12-H); 13C-
NMR (100 MHz, CDCl3) dC ¼ 207.4 (C-9), 164.2 (C-2), 158.2 (C-4),
138.6 (C-6), 115.6 (C-1), 109.1 (C-3), 111.1 (C-5), 46.1 (C-10), 25.0
(C-8), 18.6 (C-11), 14.0 (C-12), 7.7 (C-7); HRMS for C12H17O3
calcd: 209.1178; found: 209.1176 [M]H+.
1-Nitro-2,4-dihydroxy-3,6-dimethylbenzene 22. The mono-
nitration of 2,5-dimethylresorcinol was achieved with
a
mixture of diluted nitric acid and acetic anhydride at room
temperature.10,11 TLC and LC-MS was used to monitor the
reaction until it reached 70% completion. The mixture was
further diluted and extracted with ethyl acetate. The crude
extract was dissolved in methanol and puried by mass-directed
LC-MS.
ꢁ
Yellow amorphous solid; 70% yield; m.p. 130 C IR (neat):
nmax 3440, 1625, 1604, 1340 and 1194; UV (CH3CN/H2O) lmax 344
nm; 1H-NMR (500 MHz, CD3CN) dH ¼ 6.38 (s, 1H, 6-H), 2.51
(s, 3H, 8-H), 2.05 (3H, s, 7-H); 13C-NMR (125 MHz, CD3CN)
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dC ¼ 161.7 (C-1), 157.3 (C-3), 137.3 (C-5), 130.0 (C-4), 112.4 (C-6), increased accordingly for good separation. Before the run
111.4 (C-2), 23.1 (C-8), 8.4 (C-7). ended, 2 min wash of 95% organic solvent CH3CN (B) or MeOH
1-Nitroso-2,4-dihydroxy-3,6-dimethylbenzene 23 (ref. 13). The (C) was applied to ensure the elution of all compounds. Finally,
mono-nitrosation of 2,5-dimethylresorcinol was achieved using the HPLC column was prepared for the next run by adding 2
diluted sulphuric acid and sodium nitrite.12 Diluted sulphuric min 95% Water at the end of the programme.
acid (1 ml H2SO4 in 40 ml water) was added drop wise over a
vigorously stirred solution of 2,5-dimethylresorcinol (138 mg, 1
Recombinant protein overproduction and purication
mmol) and sodium nitrite (140 mg, 2 mmol) in 10 ml aqueous
Recombinant hexa-histidine tagged TropB produced by over-
ethanol 50%. The formation was monitored using TLC and LC-
expression in BL21-CodonPlus (DE3)-RP (Stratagene) using the
MS during the addition of diluted acid until the reaction
plasmid pET-28a-TropB originally described by Davison and co-
workers.4 Overproduction and purication of the protein was
according to established protocols with the exception of the
inclusion of 10% glycerol in all purication buffers and the use
of a HiLoad 16/60 Superdex 200 column (GE Healthcare) for size
exclusion chromatography (see ESI Fig. S1†).
reached to 80% completion. The reaction was diluted with 300
ml water and extracted by ethyl acetate. The crude extract was
dissolved in methanol and the compounds puried by mass-
directed LC-MS.
Red amorphous solid; 80% yield; m.p. 164 ꢁC; IR (neat): nmax
3200, 2925, 1623, 1407, 1321, 1137, 1082 and 874; UV (CH3CN/
1
H2O) lmax 210 and 303 nm; H-NMR (500 MHz, CD3CN) dH
¼
6.38 (s, 1H, 6-H), 2.35 (s, 3H, 8-H), 1.85 (3H, s, 7-H); 13C-NMR Protein Identication from SDS-PAGE gels by MALDI
(125 MHz, CD3CN) dC ¼ 171.1 (C-1), 169.8 (C-3), 150.1 (C-4),
The appropriate coomassie stained band was excised from the
SDS-PAGE gel, cut into 1 mm cubes and destained overnight
with 50% ethanol. The sample was washed with 100 mM
ammonium bicarbonate and dehydrated several times with
acetonitrile. Reduction and alkylation was achieved by incuba-
tion with 50 mM DTT at 56 ꢁC for 1 hour followed by cooling to
room temperature and incubation with 50 mM iodoacetamide
143.2 (C-5), 119.0 (C-6), 111.5 (C-2), 17.0 (C-8), 6.5 (C-7).
(ꢀ)-(Z)-2-Hydroxy-6-(hydroxyimino)-2,5-dimethylcyclohex-4-ene-
1,3-dione 28. The assay was repeated at larger scale (16 ml) with
2.5 mM of substrate. Aer complete conversion of the substrate,
the reaction mixture was acidied, claried by centrifugation
and puried directly by mass-directed LC/MS.
Colourless oil, a[D] ꢀ48.5, (c 0.35, MeOH); UV (CH3CN/H2O)
for 45 minutes in the dark. Gel pieces were washed with 100 mM
1
lmax 274 nm; H-NMR (500 MHz, CD3CN) dH ¼ 8.06 (s, N-OH),
ammonium bicarbonate and dehydrated with acetonitrile
4
4
6.28 (d, J ¼ 1.2, 1H, 4-H), 2.16 (d, J ¼ 1.2, 3H, 8-H), 1.47 (s,
3H, 7-H); 13C-NMR (125 MHz, CD3CN) dc ¼ 196.5 (C-3), 192.8 (C-
1), 152.8 (C-5), 149.4 (C-6), 127.9 (C-4), 86.0 (C-2), 25.8 (C-7), 18.2
(C-8); HRMS for C8H9NaNO4 calcd: 206.04249; found: 206.04237
[M]Na+.
several times before the addition of 12.5 ng mlꢀ1 trypsin solution
(Promega) in 50 mM ammonium bicarbonate and incubated at
37 ꢁC overnight. Peptide fragments were extracted from the gel
by several rounds of sonication in acetonitrile using a water
bath sonicator. The solution was dried overnight under vacuum
and resuspended in 10 ml of 50% acetonitrile.
Samples were spotted onto a stainless steel plate with an
equal volume of matrix (a saturated solution of a-cyano-4-
hydroxycinnaminic acid in 50% acetonitrile) and le to dry. A
mass spectrum was recorded with a MALDI-TOF spectrometer
(4700 Proteomics Analyzer, Applied Biosystems) in linear mode.
The peptides masses resulting from this analysis were
submitted to the Mascot Peptide Mass Fingerprint search
engine (http://www.matrixscience.com; Matrix Science, Lon-
don, UK) to search for matching proteins in NCBInr database
(see ESI Fig. S2†).
Preparative LC-MS
The purication of compounds was achieved using a Waters
mass-directed autopurication system comprising of a Waters
2767 autosampler, Waters 2545 pump system, a Phenomenex
˚
LUNA column (5m, C18, 100 A, 10 ꢃ 250 mm) equipped with a
˚
Phenomenex Security Guard precolumn (Luna C5 300 A)
running at 4 ml minꢀ1 and a larger column reversed-phase
˚
HPLC (Phenomenex, Kinetex, 5 mm C18 100 A, AXIA Packed LC
Column 250 ꢃ 21.2 mm) with a ow rate of 16 ml minꢀ1
.
Solvents were HPLC grade including H2O, CH3CN and MeOH
with the addition of 0.05% formic acid in all the solvent. The
post-column ow was split (100 : 1) and the minority ow was
LC-MS activity assay
made up with MeOH + 0.045% formic acid to 1 ml minꢀ1 for Enzyme activity was measured in a 1 ml nal volume with 50
simultaneous analysis by diode array (Waters 2998), evaporative mM phosphate buffer pH 8, 2.5 mM for each individual
light scattering (Waters 2424) and ESI mass spectrometry in compound and 20 mg mlꢀ1 (0.38 mM) TropB. The reaction was
positive and negative modes (Waters Quatro Micro). Detected initiated by adding 2.5 mg of NADPH (nal concentration 3
peaks were collected into glass test tubes. Combined tubes were mM) and was incubated for 60 minutes under aerobic condi-
evaporated under a ow of dry N2 gas, weighed, and residues tions at 30 ꢁC. The reaction product was extracted by ethyl-
dissolved directly in deuterated solvents for NMR analysis. acetate and detected by LC-MS. A Phenomenex LUNA column (5
˚
Solvent gradients for LC/MS analytical and purication were m, C18, 100 A, 4.6 ꢃ 250 mm) was used with a gradient elution of
variable but generally runs of 15, 30 and 60 min were designed acetonitrile: 0 min, 10% B; 15 min, 90% B; 16 min, 95% B; 17
to start with 95% Water (A) for 2 min to remove any salts present min, 95% B; 18 min, 10% B; 20 min, 10% B. Mass spectrometry
in a sample. Next, the polarity of the solvent system was was performed with a Waters ZQ micromass spectrometer.
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Control reactions were carried out under identical conditions template structure.17 This enzyme shares 25% sequence identity
but using boiled enzyme.
to TropB and is a known FAD dependent hydroxylase. The
resulting model was structurally aligned with the template 4bjy
in Pymol (The PyMOL Molecular Graphics System, Version 1.3
Determination of optimum temperature and pH
¨
Schrodinger, LLC.) and the 3D co-ordinates of the FAD ligand
Enzyme activity was measured across a range of 5 to 40 ꢁC in 50
mM Tris–HCl buffer (pH 8.0) at a xed concentration of enzyme.
NADPH (100 mM) and the natural substrate 3-methyl-
orcinaldehyde (200 mM) were included in each reaction mix.
Reactions were performed in a 100 ml volume in transparent 96
well microplates (Costar). Activity was measured by monitoring
the initial rate of increase of product formation at 400 nm in a
Molecular Devices SpectraMAX 190 microplate reader. Known
concentrations of product (dienone) were used to generate a
standard curve from which the increase in absorbance could be
converted into an increase of product concentration. The reac-
tion rate was recorded from the linear portion of a graph
showing product concentration against time. To ensure the
reactions occurred at the required temperature, solutions were
pre-incubated at that temperature for 30 minutes in either a
heat block or cooling shaker. All reactions were repeated in
triplicate.
were merged from 4bjy into the homology model le. The
resulting protein-ligand complex was subjected to energy min-
imisation using the YASARA energy minimization server.24 The
substrate, 3-methylorcinaldehyde 1 was manually positioned
within the TropB active site using Pymol and the resulting
protein-ligand complex again subjected to energy minimization
using the YASARA energy minimization server. To evaluate
TropB substrate tolerability 3-methylorcinaldehyde
1 was
substituted for a representative selection of compounds listed
in Table 1 and their ability to be accommodated with the TropB
active site assessed using the same methodology. The electro-
static surface was generated using the PDB2PQR server25 and
the APBS Pymol Plugin.26
Acknowledgements
To measure the effect of pH on activity, the above protocol
was used except temperature was xed at 30 C and different
reaction buffers were used to create a pH range of 5–12. The
following buffers were used: 50 mM sodium acetate pH 5.0,
sodium phosphate pH 6.0, Tris–HCl pH 7.0, Tris–HCl-8.0, Tris–
HCl-9.0, CHES pH 10.0 and CHES pH 12.0.
PRR is the recipient of a Royal Society University Research
Fellowship. AA thanks the Ministry of Higher Education
(mission sector-Egypt) for a Channel studentship; AAF thanks Al
Baha University, Saudi Arabia for funding. We thank EPSRC for
LCMS equipment (EP/F066104/1). AS was funded by BBSRC (BB/
I003355/1).
ꢁ
Determination of kinetic parameters
References
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value was used to calculate the extinction coefficient using the
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