Investigation of acid-base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry

Article abstract of DOI:10.1039/b821559f

The extradiol and intradiol catechol dioxygenase reaction mechanisms proceed via a common proximal hydroperoxide intermediate, which is processed via different Criegee 1,2-rearrangements. An R215W mutant of extradiol dioxygenase MhpB, able to produce a mi

Full text of DOI:10.1039/b821559f

View Online / Journal Homepage / Table of Contents for this issue
PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Investigation of acid–base catalysis in the extradiol and intradiol catechol
dioxygenase reactions using a broad specificity mutant enzyme and
model chemistry
Michela Brivio, Janne Schlosrich, Mark Ahmad, Caroline Tolond and Timothy D. H. Bugg*
Received 2nd December 2008, Accepted 18th December 2008
First published as an Advance Article on the web 9th February 2009
DOI: 10.1039/b821559f
The extradiol and intradiol catechol dioxygenase reaction mechanisms proceed via a common proximal
hydroperoxide intermediate, which is processed via different Criegee 1,2-rearrangements. An R215W
mutant of extradiol dioxygenase MhpB, able to produce a mixture of extradiol and intradiol cleavage
products, was analysed at pH 5.2–8.6, and the yield of extradiol product was found to be highly
pH-dependent, whereas the yield of intradiol product was pH-independent. The acid–base chemistry of
a biomimetic reaction for extradiol oxidative catechol cleavage was also investigated, using
1
,4,7-triazacyclononane, FeCl
a general base and (in protonated form) a general acid. Kinetic experiments using a range of meta- and
para-substituted pyridines gave a Brønsted plot of log(v) vs. pK showing a bell-shaped plot. Oxidative
catechol cleavage by a pyridine-monosubstituted b-cyclodextrin in the presence of TACN and FeCl in
2
, and pyridine in methanol, in which pyridine is proposed to act as both
a
2
methanol yielded only intradiol cleavage products. It is therefore proposed that bifunctional acid–base
catalysis is required for iron (II)-dependent extradiol catechol cleavage, whereas the rate-determining
step for intradiol catechol cleavage does not involve acid–base catalysis.
Introduction
has been found in a number of non-heme iron (II)-dependent
oxygenase enzymes.
8
The catechol dioxygenases are non-heme iron-dependent metallo–
enzymes which catalyse the oxidative cleavage of catechol sub-
strates (see Fig. 1), on bacterial catabolic pathways used for
Mechanistic studies have established that the reaction mecha-
nism of the extradiol catechol dioxygenases proceeds via a Criegee
rearrangement of a proximal hydroperoxide reaction intermedi-
1
the degradation of aromatic compounds in soil. The intradiol
9,10
ate, to give an unsaturated a-keto lactone intermediate.
The
catechol dioxygenases utilise a non-heme iron (III) cofactor to
effect cleavage of the carbon–carbon bond between the two hy-
droxyl groups, yielding muconic acid products; while the extradiol
dioxygenases utilise a non-heme iron (II) cofactor to effect cleavage
intermediacy of a proximal hydroperoxide in both extradiol and
intradiol dioxygenase mechanisms implies that these two families
of enzyme follow divergent catalytic mechanisms, in which the
choice of reaction product is dictated by the choice of alkenyl vs.
1
of the carbon–carbon bond adjacent to the two hydroxyl groups.
10
acyl migration of a common intermediate.
Crystallographic studies have shown that the active site iron (III)
cofactor of the intradiol dioxygenases is ligated by two tyrosine
The factors that control the choice of extradiol vs. intradiol re-
action pathways are therefore of interest. Studies on a biomimetic
model reaction for extradiol oxidative cleavage involving 1,4,7-
2,3
residues and two histidine residues, while the iron (II) cofactor
of the extradiol dioxygenases: is ligated by two histidine residues
triazacyclononane (TACN), FeCl
2
or FeCl , and pyridine, in
3
4–7
and one glutamic acid. The latter tridentate His,His,Glu motif
methanol, have revealed that pyridine/pyridinium ion acts as both
11
base and acid during the reaction. Site-directed mutagenesis
studies on Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-
dioxygenase (MhpB) have established that His-115 and His-
12
1
79 act as acid and base groups respectively for catalysis.
A
site-directed mutant of His-200 in Brevibacterium fuscum 3,4-
dihydroxyphenylacetate 2,3-dioxygenase by Phe gave a mutant
enzyme possessing intradiol cleavage activity, also highlighting the
role of acid–base catalysis in the choice of extradiol vs. intradiol
13
selectivity.
We have recently found using mechanistic probes that intra-
diol dioxygenase catechol 1,2-dioxygenase from Acinetobacter
sp. (CatA) follows a distinct 1,2-migration reaction mechanism,
Fig. 1 Oxidative cleavage of catechol by (a) intradiol dioxygenase
to give cis,cis-muconic acid product; (b) extradiol dioxygenase to give
2
-hydroxymuconate semialdehyde product.
14
involving O–O homolytic cleavage, as shown in Fig. 2. We
therefore wished to investigate whether the extradiol and in-
tradiol dioxygenase reactions show different acid–base catalytic
behaviour.
Department of Chemistry, University of Warwick, Coventry, CV4 7AL,
U.K.. E-mail: T.D.Bugg@warwick.ac.uk
1
368 | Org. Biomol. Chem., 2009, 7, 1368–1373
This journal is © The Royal Society of Chemistry 2009

View Online
Fig. 2 Proposed mechanisms for 1,2-rearrangement of a common hydroperoxide intermediate in the extradiol and intradiol catechol dioxygenases.
Using directed evolution, we have previously isolated two
point mutants of MhpB that produce mixtures of extradiol and
15
intradiol products: R215W (6 : 1 extradiol/intradiol) and K273R
20 : 1 extradiol/intradiol). With these mutants it is possible to
(
compare directly the partitioning of extradiol vs. intradiol product
formation at the same active site. In this study, we compare the
acid–base chemistry of extradiol vs. intradiol cleavage, using the
R215W mutant enzyme, and using the TACN extradiol model
reaction.
Results
Extradiol vs. intradiol product ratios of R215W mutant
In order to study the extradiol vs. intradiol product ratio under
different conditions, a C18 reverse phase HPLC method was
developed to separate the extradiol product 2-hydroxymuconate
semialdehyde (23.8 min) from the intradiol product cis,cis-
muconic acid (25.6 min) and remaining substrate catechol
Fig. 3 Extradiol vs. intradiol product distribution for R215W mutant of
extradiol dioxygenase MhpB at pH 5.2–8.6.
(
25.2 min). A series of incubations was carried out using mutant
R215W, in fixed pH buffers ranging from pH 5.2–8.6, and the
reaction quenched after 15 min and the product mixture analysed
using the above HPLC method.
N(CH
3
) ), and imidazole, were tested as bases in the TACN-
2
catalysed extradiol cleavage reaction, shown in Fig. 4. Kinetic
assays were carried out using 5 mM TACN, 5 mM iron (II) chloride,
The results, shown in Fig. 3, show that the formation of extradiol
product is highly pH-dependent, showing acid and base catalysis,
with maximum extradiol product formation at pH 7.0. In contrast,
the formation of intradiol product is independent of pH, in the
range pH 5.2–7.4, with a only a slight increase at pH 8.0–8.6.
The results therefore show a clear difference in acid–base catalysis
between extradiol and intradiol reaction pathways. The molar
ratio of extradiol : intradiol products at pH 8.0, calculated using
the relative extinction coefficients, is 3.4 : 1, compared with the
5
2
mM catechol, and 15 mM substituted pyridine, in methanol, at
5 C, monitoring at 316 nm. The measured rates were plotted
◦
against pK
Introduction of an electron-withdrawing group on the pyridine
ring, giving a lower pK , led to loss of activity, while introduction
of a mildly electron-donating group gave increased activity.
Highest activity was observed using 4-methoxypyridine (pK
a
, as shown in Fig. 5A, giving a bell-shaped plot.
a
a
6.47), showing 2.5- fold higher rate than with pyridine.
6
: 1 ratio measured previously for the R215W mutant by NMR
15
However, bases of higher pK , namely imidazole and p-
dimethylaminopyridine, gave lower activity. It is clear that the pK
a
spectroscopy.
a
of the acid–base catalyst does affect the rate of reaction, hence
the substituted pyridine is involved in the rate-limiting step of the
reaction.
Kinetic investigation of TACN-catalysed reaction with substituted
pyridine bases
16
Since pyridine acts as acid–base catalyst in the TACN-catalysed
The plot of log(v) vs. pK
The linear upward slope from pK
free energy relationship is followed, and that general base catalysis
a
is a Brønsted plot, shown in Fig. 5B.
extradiol model reaction, the effect of changing the pK
acid–base group in this reaction can be studied.
a
of the
a
3.0–6.5 indicates that a linear
16
A series of meta-substituted pyridines (X = CH
3
, Cl, Br,
, OCH
is occurring in the rate-determining step of the reaction. The
CN, CONH
2
) and para-substituted pyridines (X = CH
3
3
,
slope b = 0.30 indicates a significant degree of C–H bond cleavage
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1368–1373 | 1369

View Online
Fig. 4 TACN-catalysed model reaction for extradiol catechol cleavage.
70 nm, and hence was not observed in the above experiment,
2
which was monitored at 316 nm.
An intramolecular model for TACN-catalysed oxidative catechol
cleavage
Since pyridine plays an essential role in the extradiol model reac-
tion, it was of interest to examine whether intramolecular catalysis
could be achieved, using models containing TACN and a pyridine
acid–base function. Previous work had found that N-substituted
11
TACN macrocycles were inactive for extradiol cleavage. At-
tempts to synthesise a C-substituted 1,4,7-triazacyclononane or
1,4,7-triazacyclodecane macrocycle containing a pendant meta-
substituted pyridine base were unsuccessful (data not shown), due
to problems with macrocycle synthesis and deprotection.
Modified cyclodextrins are well precedented to act as
17
supramolecular hosts for aromatic molecules. Therefore, as an
alternative approach, illustrated in Fig. 6, we attached a meta-
substituted pyridine base to b-cyclodextrin, with the aim of
interacting with a catechol substrate bound non-covalently to the
cyclodextrin host. Acylation of b-cyclodextrin with pyridine 3-
carboxylic acid (nicotinic acid) was carried out in water at pH 4.75,
using EDC as coupling agent. The acylated product was purified
by cation exchange, and was isolated in 19% yield. The product was
1
confirmed to be the desired monoacylated product by H NMR
+
spectroscopy and electrospray mass spectrometry (MNa 1262.5).
Fig. 5 Rate of TACN–FeCl
pyridines as acid–base catalysts. (A) Plot of rate (v) vs. pK
plot of log(v) vs. pK . pK values: pyridine, 5.23; 4-OCH
, 5.63; 3-CONH , 3.35; 3-Cl, 2.91; 3-Br, 2.84; 3-CN, 1.39;
, 9.2; imidazole, 6.95.
2
extradiol model reaction using p-substituted
. (B) Brønsted
, 6.47; 4-CH
a
a
a
3
3
,
5
4
.98; 3-CH
-N(CH
3
2
3 2
)
Fig. 6 Modified b-cyclodextrin for catechol oxidative cleavage.
1
6
in the transition state. Although the reaction is not buffered,
the use of 3 equivalents of pyridine ensures that, after substrate
UV/vis assays of the acylated cyclodextrin with TACN, cate-
chol, and iron (II) chloride in methanol indicated that a cleavage
reaction was occurring in the presence of stoichiometric or
catalytic amounts (10%) of acylated cyclodextrin. Therefore, 10 mg
scale catechol cleavage reactions were carried out at different
catalyst loadings, and the products isolated.
Incubation of 0.1 mM 1,4,7-triazacyclononane with 0.1 mM
catechol, 0.1 mM iron (II) chloride, and 0.01 mM acylated b-
cyclodextrin in methanol, in the presence of oxygen gas for 5 h,
followed by extraction of the reaction products, identified the
11
complexation and monoanion formation, there will be 2 : 1 ratio
of pyridine : pyridinium ion present in each case.
At pK >6.5, it is apparent that there is a reduction in
a
activity, although there are only two points in this region, one
of which is imidazole, structurally different to pyridine. A larger
scale conversion was carried out using DMAP as base, and the
1
product was found by H NMR spectroscopy to consist only of
the intradiol product monomethyl muconate, which absorbs at
1
370 | Org. Biomol. Chem., 2009, 7, 1368–1373
This journal is © The Royal Society of Chemistry 2009

View Online
intradiol cleavage product monomethyl muconate as the only
product. A second experiment at the same concentrations, but
for only 3 h, also gave the intradiol cleavage product, together
with 1,2-benzoquinone, and some remaining starting material. A
third experiment containing 0.1 mM acylated b-cyclodextrin, run
for 5 h, gave 1,2-benzoquinone as the major product, and the
intradiol cleavage product and starting material, in a 9 : 1 : 1
ratio. No extradiol cleavage product was detected in any of the
three runs. Therefore, the acylated cyclodextrin is able to effect
catechol cleavage, in catalytic amounts, but only to give the
intradiol cleavage product.
determining step, to an extent of b = 0.30. General base catalysis
would be involved in the formation of the catecholate monoanion
substrate prior to hydroperoxide formation, and would also be
involved in the Criegee rearrangement step (see Fig. 7).
There is a marked decrease in the rate of extradiol product
formation in the Brønsted plot at higher values of pK
has been demonstrated that DMAP yields only intradiol cleavage
product. One possible explanation of the behaviour at high pK
a
, and it
a
is the generation of catechol di-anion by stronger bases, since the
catechol mono-anion has previously been shown to be required
11
for extradiol cleavage, therefore the decrease in activity could
be due to a decrease in the concentration of the reactive catechol
monanion complex. Alternatively, there could be a change in rate
Discussion
determining step at higher values of pK .
a
From consideration of the available evidence on the catalytic
mechanisms of the intradiol and extradiol catechol dioxygenases,
it is discussed elsewhere that both reaction mechanisms appear
to converge on a common hydroperoxide intermediate, which
undergoes alkenyl migration vs. acyl migration reactions leading
The involvement of both general base and general acid catalysis
in determining enzyme product ratios (Fig. 3) would be consistent
with the rate-determining step being the Criegee rearrangement
of a hydroperoxide intermediate, for which hydroxyl group
deprotonation is concerted with the protonation of the distal
oxygen of the hydroperoxide, as shown in Fig. 2. A concerted
general base-general acid catalysed step would be consistent
with the positioning of two histidine residues in the active site
10
to the extradiol and intradiol products respectively. It is also
apparent from both model studies and enzyme studies that both
iron (II) and iron (III) can in principle catalyse extradiol or intradiol
10,11,13,15
12
cleavage reactions.
However, the factors controlling the
of the class III extradiol dioxygenases, and would give a bell-
choice of reaction pathway are not yet resolved.
shaped pH/rate profile, as observed in Breslow’s bis(imidazole)–
19
Previous studies on the TACN–extradiol model reaction have
established that the pyridine base has a two-fold role: one
equivalent is required to act as a base, and the pyridinium salt
formed in the reaction is needed to act as a proton donor, probably
cyclodextrin model for ribonuclease A. The ability of a pyridine-
modifed b-cyclodextrin to catalyse intradiol, but not extradiol,
catechol cleavage provides a further indication that intradiol
cleavage is the “default” pathway, found when concerted acid–
base catalysis is not possible.
11
to catalyse the Criegee rearrangement in the reaction mechanism.
The importance of acid–base catalysis in the extradiol dioxygenase
reaction mechanism has been confirmed by the identification of
His-115 and His-179 as catalytic acid–base residues in the E. coli
These studies also show that there is a fairly narrow range of pK
values required for extradiol cleavage activity in the model reaction
(pK 5–7 for effective catalysis). In the extradiol dioxygenase
enzyme MhpB, pH-rate studies have indicated that the catalytic
base has a pK of 6.4, which matches well the optimum pK in
this study, and that the proton donor has a pK of 8.8. The
a
a
12
MhpB-catalysed reaction. It is not certain which step is rate-
limiting in either reaction, however, in the extradiol reaction the
final lactone hydrolysis is believed to be fast, since it has not
been possible to isolate the lactone intermediate either from model
a
a
12
a
latter value is an abnormally high value for a histidine residue,
and is controlled by a particular microenvironment in the enzyme
11
12
studies or from the enzyme-catalysed reaction, under a variety
of conditions.
12
active site. The ability of an enzyme to influence pK of catalytic
a
Determination of the extradiol/intradiol product ratios for the
R215W mutant of extradiol dioxygenases clearly shows that the
production of extradiol product is highly pH-dependent, involving
acid and base catalysis, whereas the production of intradiol
product is largely pH-independent. These data are consistent
with different mechanisms for 1,2-rearrangement of the common
hydroperoxide intermediate, as proposed recently, in which the
Criegee rearrangement step of extradiol cleavage involves bifunc-
tional acid–base catalysis, whereas the 1,2-rearrangement step
residues in this way allows for much higher rates of catalysis at
pH 7–9 in the enzyme-catalysed reaction.
In conclusion, this study provides further evidence for different
catalytic mechanisms in the extradiol and intradiol catechol
dioxygenases, with the iron (II)-dependent extradiol dioxygenases
requiring bifunctional acid–base catalysis from active site amino
acid residues.
14
Experimental
for intradiol cleavage involves homolytic O–O bond cleavage.
Physical organic chemistry studies on the reaction of 1,2-quinones
with peroxybenzoic acids have also provided evidence that the
mechanism of cleavage of the O–O bond dictates the regioselec-
Substrate conversions using R215W MhpB mutant enzyme
Mutant R215W of 2,3-dihydroxyphenylpropionate 1,2-
18
tivity of the cleavage reaction.
Using the TACN extradiol model reaction, it is possible to
examine the effects of changing the pK of the acid–base catalyst.
dioxygenase (MhpB) from Escherichia coli was expressed
15
and purified as previously described. Incubations (1.0 ml total
volume) containing 0.3 mM catechol were set up in 50 mM
sodium citrate, sodium phosphate, or Tris/HCl buffers, over the
pH range 5.2–8.6, at 0.4 pH unit intervals. To each incubation
was added 50 mg R215W MhpB, freshly re-activated by 5 mM
iron (II) ammonium sulfate and 5 mM ascorbic acid, and the
a
The Brønsted plot shown in Fig. 5B indicates that the basicity
of the pyridine base directly affects the rate of extradiol product
formation, and therefore is involved in the rate-limiting step of
the reaction. There is a clear upward slope between pK
a
3.0–
◦
6
.5, indicating that general base catalysis is involved in the rate-
mixture incubated at 25 C for 15 min. Protein was removed
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1368–1373 | 1371

View Online
Fig. 7 Mechanistic scheme for extradiol dioxygenase MhpB, showing steps involving acid–base catalysis.
by centrifugation in a Millipore Microcon Ultracel YM-10 Preparation of modified b-cyclodextrin
device. Samples (100 ml) were then injected onto a Phenomenex
Luna reverse phase C18 analytical HPLC column, and eluted
b-Cyclodextrin (2.0 g, 1.76 mmol) and nicotinic acid (0.261 g,
2.1 mmol) were dissolved in water (100 ml). The pH of the
solution was adjusted to 4.75 by addition of 0.1M NaOH.
at 0.8 ml min-1 using the following gradient of H
2
O/0.05%
COOH to methanol: 0% MeOH, 10 min; 0–20% MeOH,
min; 20–50% MeOH, 5 min, 50–70% MeOH, 5 min; 70–0%
MeOH, 5 min. Peaks were detected at 255 nm Retention times:
CF
3
1
-(3-Dimethylaminopropyl)-3-ethylcarbodi-imide hydrochloride
5
(
EDC, 0.371 g, 1.94 mmol) was added, with stirring, and the pH
-
1
-1
was then adjusted to 7.0 by addition of 0.1 M NaOH. The reaction
was stirred for 16 h at room temperature. The reaction mixture was
catechol, 25.2 min; cis,cis-muconic acid (e 17 300 M cm ),
-
1
-1
2
5.6 min; 2-hydroxymuconaldehyde (e 33 000 M cm ), 23.8 min.
+
then applied to an Amberlite C6–50 (H type) cation exchange
Product formation by MhpB has previously been shown to be
20
column (10 ¥ 3 cm), washed with water (50 ml), then eluted
with 0.05M ammonia solution. Fractions containing acylated
cyclodextrin were identified by thin layer chromatography, and
were pooled and lyophilised to give 0.429 g product (19% yield).
non-linear vs. time, with loss of enzyme activity over 2–5 min,
but the final amount of product formed is proportional to enzyme
activity.
d
H
(500 MHz, D
2
O) 8.68 (1H, s, pyr H-2), 8.40 (1H, d, J = 8.1 Hz,
pyr H-6), 7.99 (1H, d, J = 8.7 Hz, pyr H-4), 7.40 (1H, m, pyr
H-5), 5.07 (1H, s), 5.03 (1H, d, J = 3.1 Hz), 3.90–3.50 (47H, m)
ppm; d (125 MHz, D O) 161.1, 144.7, 130.8, 128.4, 125.0, 104.5,
Kinetic assay of TACN extradiol cleavage reaction using
substituted pyridine bases
C
2
+
7
3.1, 72.6, 42.0, 35.7, 23.3 ppm; m/z (ES +ve) 1262.5 (MNa );
Stock solutions of 0.1 M concentration in methanol were pre-
pared for 1,4,7-triazacyclononane, iron (II) chloride, and catechol;
and 0.3M concentration in methanol for pyridine, 3-picoline,
predicted 1262.4.
4
-picoline, 3-bromopyridine, 3-chloropyridine, 3-cyanopyridine,
Preparative scale substrate conversions
nicotinamide, 4-methoxypyridine, 4-dimethylaminopyridine, and
imidazole. In a 1.0 ml cuvette, at 25 C, final concentrations of
◦
Catechol (11 mg, 0.1 mM), iron (II) chloride (20 mg,
0.1 mM), acylated b-cyclodextrin (12.4 mg, 0.01 mM) and 1,4,7-
triazacyclononane (12.9 mg, 0.1 mM) were dissolved in 500 ml
methanol, and oxygen gas was bubbled through the stirred mixture
for 3 or 5 h at room temperature. After removal of solvent at
reduced pressure, 1M HCl (2 ml) was added, and the products
extracted into diethyl ether (2 ¥ 10 ml). The combined extracts were
5
mM TACN, 5 mM FeCl
mixed in methanol, to which was added a final concentration of
mM catechol. The reaction was monitored at 316 nm for 5
2
, and 15 mM substituted pyridine were
5
minutes, and the rate of reaction measured. Assays were carried
out in triplicate. Product formation has previously been shown to
be linear over 15 min.
10,11
1
372 | Org. Biomol. Chem., 2009, 7, 1368–1373
This journal is © The Royal Society of Chemistry 2009

View Online
dried (Na
2
SO
4
), and solvent removed at reduced pressure. The
6 K. Sugimoto, T. Senda, H. Aoshima, E. Masai, M. Fukuda and Y.
Mitsui, Structure, 1999, 7, 953–965.
reaction was also repeated at 0.1 mM concentration of acylated
b-cyclodextrin. The intradiol product monomethyl muconate was
identified by H NMR spectroscopy, as previously described.
Data for 1,2-benzoquinone: d (300 MHz, CDCl ) 7.75 (2H, t,
7
G. P. Titus, H. A. Mueller, J. Burgner and D. E. Timm, Nat. Struct.
Biol., 2000, 7, 542.
1
11,15
8 E. L. Hegg and L. Que, Jr., Eur. J. Biochem., 1997, 250, 625.
9 J. Sanvoisin, G. J. Langley and T. D. H. Bugg, J. Am. Chem. Soc., 1995,
H
3
1
17, 7836–7837.
J = 7.0 Hz), 7.55 (2H, t, J = 7.0 Hz) ppm.
1
1
0 T.D.H. Bugg and G. Lin, Chem. Commun., 2001, 941–952.
1 G. Lin, G. Reid and T. D. H. Bugg, J. Am. Chem. Soc., 2001, 123,
5
030–5039.
Acknowledgements
1
2 S. Mendel, A. Arndt and T. D. H. Bugg, Biochemistry, 2004, 43, 13390–
3396.
3 S. L. Groce and J. D. Lipscomb, J. Am. Chem. Soc., 2003, 125, 11780–
1781.
14 M. Xin and T. D. H. Bugg, J. Am. Chem. Soc., 2008, 130, 10422–
0430.
5 J. Schlosrich, K. L. Eley, P. J. Crowley and T. D. H. Bugg, ChemBiochem,
006, 7, 1899–1908.
1
We thank the ERASMUS exchange programme for financial
support (M.B.), and Dr. David Fox (University of Warwick) for
helpful discussions.
1
1
1
1
1
2
References
6 E. V. Anslyn and D. A. Dougherty, “Modern Physical Organic
Chemistry”, 2006, University Science Books, Sausalito, California, pp.
516–523.
1
2
T. D. H. Bugg, Tetrahedron, 2003, 59, 7075–7101.
D. H. Ohlendorf, J. D. Lipscomb and P. C. Weber, Nature, 1988, 336,
4
03.
17 R. Breslow, Acc. Chem. Res., 1995, 28, 146–153.
18 Y. Hitomi, H. Yoshida, T. Tanaka and T. Funabiki, J. Mol. Catal., A,
2006, 251, 239–245.
3
4
M. W. Vetting and D. H. Ohlendorf, Structure, 2000, 8, 429.
S. Han, L. D. Eltis, K. N. Timmis, S. W. Muchmore and J. T. Bolin,
Science, 1995, 270, 976–980.
A. Kita, S. I. Kita, I. Fujisawa, K. Inaka, T. Ishida, K. Horiike, M.
Nozaki and K. Miki, Structure, 1998, 7, 25.
19 E. Anslyn and R. Breslow, J. Am. Chem. Soc., 1989, 111, 5972–
5
5973.
20 T. D. H. Bugg, Biochim. Biophys. Acta, 1993, 1202, 258–264.
This journal is © The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1368–1373 | 1373