Converting phenylacetone monooxygenase into phenylcyclohexanone monooxygenase by rational design: Towards practical Baeyer-Villiger monooxygenases

Article abstract of DOI:10.1002/adsc.200505069

A homology model of the most frequently used, but thermally somewhat labile, Baeyer-Villiger monooxygenase, cyclohexanone monooxygenase (CHMO) has been derived on the basis of the recently published crystal structure of the thermally stable phenylacetone monooxygenase (PAMO). This has led to the identification of a structural element crucial for substrate acceptance and stereoselectivity, namely an arginine-interacting loop near the active site. A bulge in this loop occurring in PAMO (but not in CHMO) has been eliminated by mutation, enhancing the range of substrate acceptance and enantioselectivity of Baeyer-Villiger reactions while maintaining high thermal stability.

Full text of DOI:10.1002/adsc.200505069

FULL PAPERS
Converting Phenylacetone Monooxygenase into
Phenylcyclohexanone Monooxygenase by Rational Design:
Towards Practical Baeyer--Villiger Monooxygenases
a
a
a
a
b
Marco Bocola, Frank Schulz, FranÅois Leca, Andreas Vogel, Marco W. Fraaije,
a,
Manfred T. Reetz *
Max-Planck-Institut fꢀr Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mꢀlheim/Ruhr, Germany
a
Fax: (þ49)-208-306-2985, e-mail: reetz@mpi-muelheim.mpg.de
b
Rijksuniversiteit Groningen, Department of Biochemistry, Nijenborgh 4, 9747 AG Groningen, The Netherlands
E-mail: m.w.Fraaije@rug.nl
Received: February 1, 2005; Accepted: March 21, 2005
Abstract: A homology model of the most frequently bulge in this loop occurring in PAMO (but not in
used, but thermally somewhat labile, Baeyer–Villiger CHMO) has been eliminated by mutation, enhancing
monooxygenase, cyclohexanone monooxygenase the range of substrate acceptance and enantioselectiv-
(
CHMO) has been derived on the basis of the recent- ity of Baeyer–Villiger reactions while maintaining
ly published crystal structure of the thermally stable high thermal stability.
phenylacetone monooxygenase (PAMO). This has
led to the identification of a structural element crucial Keywords: asymmetric catalysis; enzymes; molecular
for substrate acceptance and stereoselectivity, namely modeling; oxidation; protein engineering; protein
an arginine-interacting loop near the active site. A structures
[
3,4]
Introduction
prochiral substrates (often>90% ee). Of course, sub-
strate acceptance and enantioselectivity are by nature
The Baeyer–Villiger (BV) reaction of ketones with for- limited. For this reason we recently extended the syn-
mation of the corresponding esters or lactones is an im- thetic value of this biocatalytic process by applying the
[
1]
portant transformation in synthetic organic chemistry.
methods of directed evolution in order to control the de-
[5]
It can be catalyzed by acids, bases, metal complexes or gree and direction of enantioselectivity. In that study
flavin-dependent enzymes. The latter are generally whole-cell preparations of E. coli expressing the re-
[
2]
[6]
called Baeyer–Villiger monooxygenases (BVMOs),
combinant CHMO from Acinetobacter sp. NCIMB
[5]
an example of which are the cyclohexanone monooxy- 9871 were used. However, due to the fact that at that
[
2,3]
genases (CHMOs). It has been shown that in this en- time not a single BVMO had been structurally charac-
zymatic process dioxygen (air) reacts with the reduced terized by X-ray crystallography, it was not possible to
enzyme-bound flavin (FAD) to form an intermediate relate the results to a molecular basis.
peroxide-anion which, in the deprotonated form, ini-
Most recently, Malito, Alfieri, Fraaije and Mattevi re-
[7]
tiates the BV reaction by nucleophilic addition to the ported the first crystal structure of a BVMO. The so-
[
2,3]
carbonyl function. This process leads to the so-called called phenylacetone monooxygenase (PAMO) from
Criegee intermediate which then rearranges to the ester the moderately thermophilic bacterium Thermobifida
[8]
(
or lactone). Ultimately O delivers one oxygen atom to fusca, discovered previously by genome mining, was
2
the substrate, the other being reduced to water (Scheme shown to exhibit a two-domain architecture resembling
). The oxidized flavin then needs to be recycled by re- structural characteristics of disulfide oxidoreductases.
1
duction, the coenzyme NADPH taking over this func- The crystal structure reveals a cleft at the domain inter-
tion. Therefore, when employing the isolated CHMOs, face, at which the active site is located. Arginine at posi-
NADPH itself has to be recycled. For this reason organic tion 337 was identified above the bound flavin ring in a
chemists have found it more convenient to use whole position ideally suited to stabilize the negatively charg-
[2–4]
[7]
cells.
Employing CHMO-containing strains from ed flavin peroxide. Two further points deserve men-
various organisms, this procedure has been applied suc- tion: Firstly, this BVMO is the thermally most robust
cessfully in the kinetic resolution of a variety of different of its kind known to date, making it an attractive candi-
chiral ketones and in the desymmetrization of various date for potential application in organic chemistry. Sec-
Adv. Synth. Catal. 2005, 347, 979–986
DOI: 10.1002/adsc.200505069
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Marco Bocola et al.
Scheme 1. Schematic representation of BVMO-catalyzed Baeyer–Villiger reactions of ketones.
ondly, of various ketone substrates tested in the BV re- Results and Discussion
action, phenylacetone was shown to be oxidized most
[8]
rapidly. PAMO appears to prefer substrates bearing
phenyl groups, although it is currently not possible to de-
fine the pocket in which the aromatic moiety binds. In
Homology Model of CHMO
contrast, ketones such as cyclohexanone are not accept- In order to generate a structural model of CHMO based
[
8]
ed or react extremely slowly. Thus, PAMO has the at- on the crystal structure of PAMO (pdb-code 1W4X), we
tractive feature of being unusually stable, but its range of applied the CBS-CPHmodels-2.0 3D-homology model-
[
9]
substrate acceptance is rather limited and enantioselec- ing server. The sequence identity of the CHMO (523
[8]
tivity is low.
amino acids) and PAMO (524 amino acids) is 40.3%,
In Mꢀlheim we became interested in the crystal struc- and the calculated BLAST-alignment score is 526. Fig-
ture of PAMO because it allowed us to construct a struc- ure 1 shows a portion of the CHMO/PAMO alignment
tural model of the wild-type (WT) CHMO and the high- focusing on the differences in the arginine-interacting
ly enantioselective mutants that we had created earlier loops of the two enzymes.
[
5]
by directed evolution. Moreover, another point of in-
All conserved motifs typical of flavin and nicotin-
terest emerged, which is the subject of the present study. amide cofactor binding sites and other known sequence
[
10]
Upon comparing the CHMO homology model with the motifs for monooxygenases
are correctly aligned,
crystal structure of PAMO, a remarkable structural dif- which speaks for the reliability of the predicted 3D mod-
ference became apparent, namely the presence of a el. Figure 2 (top) shows the overall fold of PAMO and
bulge in the loop near the active site of PAMO which the CHMO homology model. As already mentioned in
is absent in CHMO (see below). One can imagine that the introduction, the most prominent structural differ-
the presence of this bulge places limits on the range of ence between PAMO and CHMO is the presence of
substrate acceptance of PAMO. Therefore, full or partial an additional bulge in one of the loops of PAMO. The
elimination of the bulge could turn the PAMO into an bulge is defined by Ser-441, Ala-442 and Leu-443 (com-
enzyme variant displaying an enlarged substrate accept- pare Figure 1). This loop occurs in the FAD-binding do-
ance while maintaining its thermal stability. This in- main and is spatially located next to the catalytic argi-
spired us to perform “rational” engineering of PAMO nine (Arg-337) (Figure 2, bottom). As delineated below,
with theaim ofobtaininga “phenylcyclohexanone mono- Arg-337 is instrumental in stabilizing the negatively
oxygenase” (PCHMO). Indeed, 2-phenylcyclohexa- charged flavin peroxide intermediate. Our previous
none is a substrate that is hardly accepted by the WT work concerning the directed evolution of enantioselec-
PAMO.
tive BV monooxygenases showed that position 432 of
CHMO is one of the important hot spots, and we now
Figure 1. Differences in the arginine-interacting loop between PAMO and CHMO. Excerpt of the sequence alignment be-
tween PAMO and CHMO. Identical residues are shaded black and homologous residues are in gray. The blue box surrounds
the arginine-interacting loop. The amino acids that were mutated in this study are colored red.
980
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Practical Baeyer–Villiger Monooxygenases
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Figure 2. Comparison of the crystal structure 1W4X of PAMO (left) and the homology model of CHMO (right) with 40.3%
sequence identity. The upper part shows the overall fold and the lower part is a zoom into the active site showing the FAD
cofactor as solid sticks and the catalytic arginine in ball-and-stick model. The yellow color highlights the presence of two addi-
tional amino acids in the arginine-stabilizing loop of PAMO compared with CHMO drawn as a backbone representation.
know that it occurs in the corresponding (shorter) argi- * P1: deletion of Ala-442,
nine-interacting loop. Mutations at this position have a * P2: deletion of Ala-442 and Leu-443, and
dramatic influence on the direction and magnitude of * P3: deletion of Ser-441 and Ala-442.
[
5]
enantioselectivity of BV reactions and sulfoxidation
[
11]
of prochiral thioethers. In our homology model of The enzyme variants were generated by Quick-Change
CHMO Phe-432 occurs at the position corresponding PCR from the pPAMO-vector described elsewhere and
to Leu-443 at the start of the bulge in PAMO (for align- expressed in E. coli TOP10-cells (see Experimental Sec-
[
8]
ment see blue box in Figure 1).
tion).
Mutational Studies at the Bulge of PAMO
Catalytic Profile of the PAMO Variants
Our immediate goal was to generate three PAMO var- We initiated the study of substrate acceptance by sub-
iants by manipulating the bulge in the arginine-interact- jecting phenylacetone (1) and 2-phenylcyclohexanone
ing loop in the following way:
(3) to the BV reaction using the purified PAMO variants
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Marco Bocola et al.
hand, suggest further interactions that cannot be readily
explained at this time.
While increasing the affinity for cyclohexanone deriv-
atives by deleting the bulge, the catalytic efficiency in
the oxidation of the parent substrate phenylacetone
(
1) is actually decreased. Although we could get high
conversions in whole-cell catalysis, the KM values for
all three generated PAMO variants are significantly in-
creased over that of the WT enzyme. Importantly, the
thermostability of the new PAMO variants is compara-
ble to that of the wild-type and was determined at
5
2
08C. For variant P1 we found a half-life time of
9 hꢀ2 h with a free energy for the denaturation of
in the presence of defined amounts of NADPH. Enzyme 105.3 kJ/molꢀ0.5 kJ/mol, for variants P2 and P3 half-
activity was determined by UV/Vis spectroscopy by life times of 16 hꢀ3 h (103.7 kJ/molꢀ0.5 kJ/mol) and
monitoring the decrease in NADPH concentration, 17 hꢀ2 h (103.9 kJ/molꢀ0.5 kJ/mol), respectively.
which is conveniently possible by measuring the de- These half-life times are comparable to those measured
crease in characteristic absorbance at 340 nm. All ex- for the wild-type enzyme.
periments were performed at 258C using air-saturated We then turned to the use of whole cells and expanded
Tris-HCl buffers according to a modified literature pro- the list of substrates to include ketones 5–8. The results
[
8]
[8]
cedure.
show that, under the conditions used, only those ketones
The steady-state kinetic parameters (Michaelis– undergo BV reactions that have a phenyl group near the
Menten) are summarized in Table 1. It can be seen carbonyl function, namely 1, 3 and 7. In contrast, cyclo-
that the catalytic profile is clearly changed by deleting hexanone (5), 4-phenylcyclohexanone (6) and proges-
the bulge from the arginine-interacting loop. A cyclo- terone (8) do not react to any appreciable extent.
hexanone derivative such as 2-phenylcyclohexanone
(
3), which is hardly turned over by the WT PAMO
with a conversion barely reaching 10% in whole-cell cat-
alysis within 24 hours (see below), shows almost full con-
version with, for example, mutant P3 as catalyst. Mutant
P3 has a K value of 0.07 mM for 2-phenylcyclohexa-
M
none (3), comparable to the K of 0.059 mM observed
M
for the WT PAMO with respect to phenylacetone
[8]
(
1). The conversion of 2-phenylcyclohexanone (3) by
mutant P2, the second PAMO variant with the size of
the arginine-positioning loop being reduced to the size
of the corresponding loop in CHMO, is comparable to
Compounds 3 and 7 are chiral, which offers the oppor-
mutant P3. Its KM towards this substrate is 0.5 mM. tunity to study kinetic resolution. Although the WT
This indicates that steric constraints applied by the argi- PAMO requires very long reaction times for reasonable
nine-interacting loop play a major role in the substrate conversion, it was included in the study. Table 2 shows
binding by the enzyme. The significant differences in the conversions and enantioselectivities obtained in
KM values between mutants P2 and P3, on the other whole-cell catalysis of ketones 3 and 7. WT PAMO oxi-
Table 1. Steady-state kinetic parameters of WT PAMO and variants.
ꢁ
1
ꢁ1 ꢁ1
Substrate
PAMO variant
KM [mM]
kcat [s
]
k /K [M
s ]
cat
M
[
a]
1
1
1
1
3
3
3
3
WT
P1
P2
0.059
1.9
32000
82
3ꢀ1
0.25ꢀ0.02
0.40ꢀ0.04
0.22ꢀ0.01
n.d.
4ꢀ2
99
89
n.d.
134
1054
3395
P3
2.5ꢀ0.4
WT
n.d.
[
b]
b]
b]
P1[
2.3ꢀ0.5
0.5ꢀ0.1
0.07ꢀ0.01
0.31ꢀ0.03
P2
P3
0.5ꢀ0.2
[
0.25ꢀ0.02
[
a]
b]
[8]
Literature value measured without addition of acetonitrile.
A racemic mixture was used.
[
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dizes ketone 3 with low conversions and essentially no
stereoselectivity, whereas we observed increasing con-
versions in the case of the mutants in the order P1
<
P2<P3. The most active PAMO variant P3 also shows
excellent stereoselectivity in the kinetic resolution of
ketone 3. An E value of 100 at 50% conversion was ob-
tained after 2 h of reaction in favor of (R)-4. Significant-
ly, the WT CHMO also shows (R) selectivity in this ki-
[
12]
netic resolution with an E-value of>100. However,
as already pointed out its thermal stability is fairly low.
Thus, mutant P3 is characterized by the thermal stability
of WT PAMO and the high enantioselectivity of WT
CHMO.
Conversion of substrate 7 is in general less efficient
than that of substrate 3. It is significantly converted
only by mutant P3. In this reaction only one enantiomer-
ic form of lactone 9 could be detected by chiral GC at a
conversion of 40% (E>200) in favor of the (R)-product,
which again is similar to the oxidation by the WT
CHMO.
Since in our previous work concerning the directed
[
13c]
evolution of CHMOs we had discovered that certain cording to a procedure described previously. This was
mutants are not only excellent catalysts for asymmetric required because we started from an experimental crys-
[5]
BV reactions, but also for enantioselective oxidations tal structure without bound ligand. This exploratory
[
11]
of prochiral thioethers, we decided to test the PAMO modeling shows that a difference in the arginine-inter-
[
4f–m]
variants in the sulfoxidation of thioether 10.
PAMO acting loop between PAMO and CHMO, namely the
variant P3 turned out to be quite enantioselective, lead- bulge defined by Ser-441, Ala-442 and Leu-443 in
ing to an ee value of 90% in favor of the (R)-11. For com- PAMO, sterically constrains Arg-337 and thus prevents
parison, WT PAMO and CHMO give an ee value of conversion of cyclic ketones.
7
9.5% and 14%, respectively.
We then modeled the substrate 3 into the PAMO var-
iant P3 in which Ser-441 and Ala-442 are deleted. Fig-
ure 3 shows that a shortening of this active site loop by
two amino acids leads to a PAMO variant which is
able to bind 2-phenylcyclohexanone (3) in the form of
Molecular Modeling of the Criegee Intermediate
We compared the structure of the active sites and ana- a Criegee intermediate, the latter being stabilized by
lyzed the differences by modeling the Criegee inter- H-bonding to Arg-337 (Figure 3). In sharp contrast,
mediate arising from the reaction of 2-phenylcyclohexa- the WT PAMO does not provide enough space in the ac-
none (3) into the PAMO crystal structure and the creat- tive site to position substrate 3 in the form of a Criegee
ed CHMO homology model using the united atom force intermediate due to the presence of the bulge. Thus, we
field MAB as implemented in MOLOC with implicit propose that the nature of the arginine-interacting loop
[13]
solvation.
The structures were subsequently mini- (Figure 2) is responsible for the different substrate rec-
mized and relaxed by molecular dynamics to analyze ognition when going from linear ketones like phenylace-
the geometry of the active site with bound substrate ac- tone (1) to cyclic ketones like 2-phenylcyclohexanone
Table 2. Kinetic resolution of ketones 3 and 7 using whole cells.
[a]
Ketone
Enzyme variant
E
% conversion after 24 h
Preferred enantiomer
3
3
3
3
7
7
7
7
WT-PAMO
P1
P2
P3
1.2
35
23
100
–
10
51
72
91
0
0
0
40
S
R
R
R
–
–
–
WT-PAMO
P1
P2
P3
–
–
>200
R
[
a]
E-values were calculated on the basis of 40–50% conversion (except for the WT).
Adv. Synth. Catal. 2005, 347, 979–986
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Marco Bocola et al.
We have shown that manipulation of this structural ele-
ment stronglyinfluences substrate acceptance and enan-
tioselectivity.
The comparison of the crystal structure of the WT
PAMO with the CHMO homology model allowed us
to rationally redesign the PAMO with the goal of ex-
panding the range of its substrate acceptance while
maintaining thermal stability. The PAMO was success-
fully turned into a phenylcyclohexanone monooxyge-
nase (PCHMO). This was accomplished by deleting ma-
jor parts of a bulge occurring in the arginine-interacting
loop near the active site. The observation that fast turn-
over requires the presence of a phenyl group near the
carbonyl function of the ketone substrate illuminates
the scope and limitation of the present PAMO variants.
This structural feature is currently difficult to explain,
and we refrain from unfounded speculations. Neverthe-
Figure 3. Model of the PAMO variant P3 with deleted Ser- less, on the practical side the developments described in
4
41 and Ala-442 containing the Criegee intermediate arising
this study can be considered to be steps towards the ul-
timate goal of creating thermostable BV monooxyge-
nases showing broad substrate acceptance and high de-
grees of enantioselectivity. One logical approach would
be to perform saturation or cassette mutagenesis in
the arginine-interacting loop. Finally, the structural
from 2-phenylcyclohexanone (3) in a reactive geometry with
two H-bonds originating from the catalytic Arg-337.
[15]
(
3). The reason for the necessity of phenyl groups near and mechanistic information emerging from the present
the carbonyl function still needs to be clarified.
study is helping us in the interpretation of the results of
our previous investigation concerning the directed evo-
[
5,11]
lution of enantioselective CHMOs.
One of the muta-
Conclusions
tions occurring at an important “hot spot” responsible
for enhanced stereoselectivity was reported to be
[
4a]
Since the early work of Taschner describing the appli- F432S, which we can now identify as being in the loop
cation of Baeyer–Villiger monooxygenases as catalysts near the active site. Ser-432 probably forms a hydrogen
in organic chemistry, a number of other synthetic organ- bond with the catalytically active arginine. More de-
ic chemists have exploited these enzymes in asymmetric tailed theoretical and experimental studies are in prog-
transformations, usually employing a CHMO in the ress.
[
2–4]
form of whole cells.
In spite of the pioneering mech-
[2]
anistic work of a number of biochemists, detailed inter-
pretations of asymmetric BV reactions have not been
possible due to the long-standing problem of obtaining
suitable crystals of these relatively unstable enzymes,
making crystallographic characterization impossible.
For this reason all published models of the stereochem-
ical outcome of enzyme-catalyzed BV reactions consti-
Experimental Section
Materials
Phenylacetone (1), 2-phenylcyclohexanone (3), cyclohexa-
none (5), 4-phenylcyclohexanone (6), 2-benzylcyclohexanone
[
3,4,14]
tute useful mnemonic devices,
but they really do
(
7) and progesterone (8) were obtained from Aldrich and Flu-
not include structural aspects on a molecular basis.
The recently published crystal structure of a thermosta-
ka. p-Methylbenzyl methyl sulfide was synthesized using a pro-
cedure described in the literature. Racemic lactones for GC
analysis were synthesized by mCPBA oxidation of the ketones.
[
7]
ble Baeyer–Villiger monooxygenase PAMO
has
changed the situation. PAMO has a relatively narrow NADPH was obtained from Jꢀlich Fine Chemicals. DNA se-
range of substrate acceptance, but it is thermally ro- quencing was done at Medigenomix (Martinsried, Germany).
[8]
bust. In the present study we have utilized the structur-
al information in order to construct a homology model
of the widely used CHMO. As a result we have been Mutation and Expression of Recombinant PAMO
able to apply molecular modeling, which has emerged
in the identification of a structural element essential in
enzyme-catalyzed BV reactions: the arginine-interact-
pPAMO was purified from E. coli TOP10 cells using the QIA-
prepr Spin Miniprep Kit and subjected to Quick-Change PCR
following the Stratagene Quick-Change-Kit protocol (Strata-
ing loop, as shown in Figures 1 and 3. The presence of gene) with HotStart KOD-Polymerase (Novagen) using the
this loop seems to be conserved throughout BVMOs. following primers:
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P1-fw: 5’-aggcccgggcagcccgtctctcagcaacatgctggtct-3’, P1-
rev: complementary to P1-fw,
P2-fw: 5’-aggcccgggcagcccgtctagcaacatgctggtctcta-3’, P2-
rev: complementary to P2-fw,
P3-fw: 5’-gcaggcccgggcagcccgctcagcaacatgctggtc-3’, P3-rev:
complementary to P3-fw.
The resulting plasmids were transformed into E. coli
TOP10. Integrity of the coding region was verified by DNA se-
quencing. For expression the cells were cultivated in either LB-
Medium (for whole-cell catalysis) or TB-Medium (for purifica-
tion of the PAMO variants) supplemented with 0.1% l-arabi-
nose and 100 mg/mL carbenicilline at a temperature of 378C.
the case of 2-benzylcyclohexanone (7) 1 mL of a solution of
2-hydroxypropyl-b-cyclodextrin (Wacker CAVASOL W7 HP
Pharma, 134.3 mg/mL in water) was added for solubilization.
The culture was further grown at 378C in 100-mL Erlenmeyer
flasks. Samples were taken at various time intervals and ex-
tracted with 2 equivalents of ethyl acetate and subsequently an-
alyzed by chiral GC (column and condition: 30 m BGB 177 and
BGB 15 column, respectively, 0.25 mm i.d., 0.25 mm film;
G/468; 0.5 bar hydrogen pressure, 76 min isotherm 1308C,
25 min, isotherm 2008C, 15 min). The assignment of absolute
configuration of the resulting lactones is based on literature
[16]
[12]
data. In the case of kinetic resolution, the formula of Sih
was used to obtain the E values.
Purification of Recombinant PAMO Variants
Sulfoxidation of p-Methylbenzyl Methyl Sulfide (10)
Purification of the PAMO variants was performed as described
elsewhere for WT PAMO with minor modifications.
2
0 mL of heat-treated TOP10-expression culture were mixed
with 15 mL of neat p-methylbenzyl methyl sulfide (10) and
mL of a solution of 2-hydroxypropyl-b-cyclodextrin (Wacker
[8]
3
CAVASOLW7 HP Pharma, 134.3 mg/mL in water) were add-
ed. The culture was further grown at 308C and extracted with 2
equivalents of dichloromethane and analyzed by chiral HPLC
Steady-State Kinetics
[11]
Protein concentrations were measured photometrically by
monitoring the absorption of the FAD-cofactor at 441 nm
as previously described.
ꢁ
1
ꢁ1
(
e441 ¼12.4 mM cm ). For steady-state kinetics purified
PAMO mutants at a concentration of 1 mM were dissolved in
0 mM Tris-HCl (pH 8.0) in the presence of 100 mM NADPH.
Acknowledgements
5
Substrates were dissolved in acetonitrile and added to the pre-
mixed enzyme solution resulting in an acetonitrile concentra-
tion of 2.5% (v/v). NADPH consumption was continuously
measured at 340 nm (e340 ¼6.22 mM cm ). The kinetics
were measured at 258C. The obtained data were fitted to the
Michaelis–Menten equation by non-linear regression analysis
implemented in the program Origin 7.0 (Microcal, Northamp-
ton, MA). For measuring the thermostability of the PAMO var-
iants, solutions of the purified enzymes were incubated at
We thank Silvia Ruthe and Heike Hinrichs for help with the
chromatographic work. Support by the Fonds der Chemischen
Industrie is gratefully acknowledged.
ꢁ
1
ꢁ1
References and Notes
[
1] a) G. R. Krow, Org. React. (N. Y.) 1993, 43, 251–798;
b) M. Renz, B. Meunier, Eur. J. Org. Chem. 1999, 737–
508C. Activity was determined as described above in the pres-
ence of 20 mM phenylacetone (substrate saturation condi-
tions). The data were fitted to a single exponential decay curve
using Origin 7.0 and the half-life time was calculated. The free
energy of denaturation was calculated from the deactivation
7
50; c) G. Strukul, Angew. Chem. 1998, 110, 1256–1267;
Angew. Chem. Int. Ed. 1998, 37, 1198–1209; d) C.
Bolm, T. K. K. Luong, O. Beckmann, in: Asymmetric Ox-
idation Reactions, (Ed.: T. Katsuki), University Press,
Oxford, 2001, p. 147.
rate k using the formula:
d
[
2] Isolation, characterization and/or mechanistic studies of
CHMOs as whole cells or isolated enzymes: a) G. E. Tur-
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