Titration and assignment of residues that regulate the enantioselectivity of phenylacetone monooxygenase

Article abstract of DOI:10.1002/adsc.200600598

Phenylacetone monooxygense (PAMO) from Thermobifida fusca was employed for the asymmetric oxidation of thioanisole (sulfoxidation) and of racemic 2-phenylpropionaldehyde (Baeyer-Villiger oxidation). A pH dependence of enantioselectivity was observed in both cases. Two different residues, with pK_a values of 7.8 ± 0.2 and 9.2 ± 0.2, appeared to be responsible for the pH effects on PAMO enantioselectivity. The protonation of Arg337 and the FAD:C4a-hydroperoxide/ FAD :C4a-peroxide equilibrium were identified as the major factors responsible for the fine-tuning of PAMO enantioselectivity in Baeyer-Villiger oxidation and sulfooxidation, respectively.

Full text of DOI:10.1002/adsc.200600598

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DOI: 10.1002/adsc.200600598
Titration and Assignment of Residues that Regulate the
Enantioselectivity of Phenylacetone Monooxygenase
Francesca Zambianchi,a Marco W. Fraaije,b Giacomo Carrea,a
Gonzalo de Gonzalo,c Cristina Rodríguez,c Vicente Gotor,c
and Gianluca Ottolinaa,*
a
Istituto di Chimica del Riconoscimento Molecolare, CNR, via Mario Bianco 9, 20131, Milano, Italy
Fax : (+39)-(0)22-890-1239; e-mail: gianluca.ottolina@icrm.cnr.it
Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,
b
Nijenborgh 4, 9747 AG Groningen, The Netherlands
Departamento de Química Orgµnica e Inorgµnica, Universidad de Oviedo, c/Julian Clavería, 33006, Oviedo, Spain
c
Received: November 17, 2006; Revised: February 28, 2007
Supporting information for this article is available on the WWW under http://asc.wiley-vch.de/home/.
Abstract: Phenylacetone monooxygense (PAMO)
from Thermobifida fusca was employed for the
asymmetric oxidation of thioanisole (sulfooxida-
tion) and of racemic 2-phenylpropionaldehyde
(Baeyer–Villiger oxidation). A pH dependence of
enantioselectivity was observed in both cases. Two
different residues, with pKa values of 7.8ꢀ0.2 and
9.2ꢀ0.2, appeared to be responsible for the pH ef-
fects on PAMO enantioselectivity. The protonation
of Arg337 and the FAD:C4a-hydroperoxide/
FAD:C4a-peroxide equilibrium were identified as
the major factors responsible for the fine-tuning of
PAMO enantioselectivity in Baeyer–Villiger oxida-
tion and sulfooxidation, respectively.
acidic or basic groups, which must be either charged
or uncharged during the enzyme-catalyzed reactions.
Since the protonation of these groups depends on
their pKa values, the variation of reaction pH would
modify the active site microenvironment,[1] leading
then to a variation of enantioselectivity.
Actually, a pH dependence of enantioselectivity has
been described in several cases, but these studies have
not given any indication on the residues involved in
the modification of enzyme selectivity.[3] In other
cases, models capable to correlate enantioselectivity
and kinetic constants (KM and Vmax) at different pHs
have been proposed for enzymatic transformations;
however, fitting the experimental results has been
quite cumbersome and problematic.[4]
Keywords: biocatalysis; chirality; enantioselectivity;
monooxygenase; oxidation; pH
Baeyer–Villiger monooxygenases are an interesting
class of enzymes belonging to the family of flavin-de-
pendent monooxygenases which are able to oxidize
several compounds such as ketones, organic sulfides
and amines.[5] These enzymes are gaining interest in
synthetic organic chemistry since they are able to use
In enzymatic reactions the proper protonation state environmentally benign molecular oxygen to catalyze
of catalytically important residues is required for effi- the oxidation of substrates in an asymmetric
cient catalysis to occur.[1] The role of these residues manner.[6] Numerous microorganisms produce these
has been unveiled by, inter alia, structural studies enzymes, and amid them, the moderate thermophilic
combined with site-directed mutagenesis experiments. bacterium Thermobifida fusca expresses an appealing
As an example, by means of NMR experiments, the Baeyer–Villiger monooxygenase: phenylacetone mon-
histidine residue present in the catalytic dyad His-Asp ooxygense (PAMO; EC 1.14.13.92).[7] The structure of
of glucose 6-phosphate dehydrogenase of Leuconos- this monomeric enzyme, which was recently resolved
toc mesenteroides has been clearly pinpointed and ti- (Figure 1, PDB 1W4X), displays some remarkable
trated, and its pKa value simply obtained by fitting features: i) a two-domain architecture resembling that
the chemical shift values with the Henderson–Hassel- of the disulfide oxidoreductases and ii) an arginine
bach equation.[2] The protonation state might also in- residue that lays above the flavin ring in a position
fluence the stereochemical outcome of the products. suited to stabilize the negatively charged flavin perox-
In fact, a pH dependence of stereoselectivity is con- ide and Criegee intermediates.[8]
ceivable for enzymes that have, in the active site,
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Francesca Zambianchi et al.
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Scheme 2. Enantioselective PAMO-mediated Baeyer–Villig-
er oxidation of racemic 2-phenylpropionaldehyde to (S)-
formic-acid 1-phenylethyl ester.
meric excess (ee) values increased from 9.6 to 44.9%
by raising the pH from 6 to 10 (Figure 2). The enan-
tiomeric excess values were obtained from several
Figure 1. Model of the three-dimensional structure of
PAMO (FAD and Arg337 in black).
As part of a long-term program aimed at exploiting
the use of oxidative enzymes in synthetic organic
chemistry, we recently became interested in the use of
PAMO as a catalyst for the asymmetric oxidation of
organic sulfides and ketones.[9] In the present work we
have investigated the effect of pH on the enantiose-
lectivity of PAMO, using as substrates thioanisole
Figure 2. pH dependence of enantiomeric excess (ee) of the
product (R)-methyl phenyl sulfoxide in the PAMO oxidation
of thioanisole. The pH values remained constant throughout
the entire experimental period. The correlation coefficient
of the curve was 0.996.
(methyl phenyl sulfide) (Scheme 1) and 2-phenylpro- measurements, at different degrees of conversion (5–
pionaldehyde (Scheme 2). In the oxidation of thioani- 100%). It should be mentioned that the asymmetric
sole to the corresponding (R)-sulfoxide, the enantio- oxidation of thioanisole to sulfoxide was accompanied
by the overoxidation to achiral sulfone (Scheme 1).
The second oxidation step was not enantioselective as
demonstrated by: i) the ee values of the (R)-sulfoxide
did not change as a function of conversion degree,
and ii) no kinetic resolution was observed starting
from racemic sulfoxide (see Supporting Information).
The pH profile of the ee values resembled a simple
acid/base titration curve (Figure 2) and, therefore, the
data were fitted in the acid dissociation equilibrium
equation (Henderson–Hasselbach). The pKa value
was determined by the three-parameter fit of the fol-
lowing equation:
Scheme 1. Pathway of the PAMO-catalyzed sulfoxidation of
thioanisole to methyl phenyl sulfoxide and methyl phenyl
sulfone.
eeHA þ eeAꢁ ꢂ 10pHꢁpK
a
eeobs
¼
1 þ 10pHꢁpK
a
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Residues that Regulate the Enantioselectivity of Phenylacetone Monooxygenase
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Table 1. PAMO amino acids with theoretical pKa values[a]
close to 7.8ꢀ0.2 or 9.2ꢀ0.2.
where eeHA is the enantiomeric excess in the acidic
pH regime and eeA- the enantiomeric excess at basic
pH. The fitted pKa value was found to be 7.8ꢀ0.2.
The second studied conversion refers to the
PAMO-mediated Baeyer–Villiger oxidation of race-
mic 2-phenylpropionaldehyde to (S)-formic-acid 1-
phenylethyl ester which leaves the (R)-2-phenylpro-
pionaldehyde mostly unreacted (Scheme 2). Enzyme
enantioselectivity, expressed as enantiomeric ratio,[10]
showed with the aldehyde substrate an opposite trend
with respect to the sulfide substrate as it decreased
from 26.5 to 16.7 on raising the pH from 7 to 10
(Figure 3). The data resembled again an acid/base ti-
tration curve, and, therefore the pKa was determined
as already described, giving a value of 9.2ꢀ0.2.
Amino acid
pKa
DpKa
Ca-FAD:C4a [Š]
Arg10
His137
Glu341
Cys73
Lys239
Lys336
Cys528
Arg337
Tyr56
7.72
7.53
7.35
8.31
9.29
9.29
9.34
9.36
8.86
0.08
0.27
0.45
0.51
0.09
0.09
0.14
0.16
0.34
30.2
31.8
13.0
11.2
22.4
11.0
23.7
7.5
12.2
[a]
The pKa values were calculated with the PROPKA
method[11] (see text).
(smaller DpKa) and their distance[8] from the flavin
(Ca-FAD:C4a). For the pKa 7.8ꢀ0.2, Arg10 and
His137 are too far away from the flavin (more than
30 Š); instead, Glu341 and Cys73, which are closer to
the flavin, have DpKa values that deviate by more
than double the standard deviation. Furthermore,
Cys73 is on the si side of the flavin, which is opposite
to the active site, with a distance Ca-FAD:C4a as high
as 11.2 Š. Moreover, the side chain of this amino acid
is pointing away from the FAD:C4a, with a sulfur-
FAD :C4a distance of 13.0 Š.[8] The data suggest that
the variation of enzyme enantioselectivity in the oxi-
dation of thioanisole cannot be linked to a specific
protonation state of an amino acid close to the active
site. However, it should be emphasized that there is
another functional group in the active site that can be
influenced by variations of pH, that is, the FAD co-
factor or, more precisely, the FAD:C4a-hydroperox-
ide/FAD:C4a-peroxide derivatives. For the closely re-
lated enzyme cyclohexanone monooxygenase from
Figure 3. pH dependence of enantioselectivity, expressed as
enantiomeric ratio of PAMO in the oxidation of 2-phenyl-
propionaldehyde. The pH values remained constant
throughout the entire experimental period. The correlation
coefficient of the curve was 0.986.
Although the two reactions were performed in the Acinetobacter sp. NCIB9871, these two intermediates
same conditions, two different residues seem to be re- can be interconverted by changing the pH. The inter-
sponsible for the pH effects on PAMO enantioselec- conversion showed a pKa of 8.4ꢀ0.2.[13] This value is
tivity in the oxidation of an electron-rich substrate quite close to pKa 7.8ꢀ0.2. Therefore, it is tempting
(sulfide) or an electron-poor substrate (aldehyde).
to conclude that the observed pKa of PAMO related
Nowadays it is possible to predict with good ap- to enantioselectivity in sulfoxidations reflects the pKa
proximation the pKa of every amino acid in proteins of the PAMO FAD:C4a-hydroperoxide/FAD:C4a-
and, among the many methodologies that have been peroxide equilibrium.
proposed, we have chosen the PROPKA method to
For the observed pKa of 9.2ꢀ0.2, Arg337, which
calculate pKa values.[11] A survey on mutations that in- has pKa of 9.36 and is only at 7.53 Š from the flavin,
fluence enzyme enantioselectivity has revealed that in appears of special interest (Table 1). All other amino
most cases mutations closer to the active site are acids (Lys239, Lys336, Cys528 and Tyr56), are too far
more effective than distant ones.[12] Mutations distant from the active site and, thus, unable to influence
more than 10 Š (Ca) from the key active site atom enzyme selectivity. In PAMO, Arg337 is indispensable
are generally poorly effective in perturbing enantiose- for catalysis as demonstrated by the finding that
lectivity. The same should hold for the protonation Arg337Ala and Arg337Lys replacements completely
states of amino acids: the closer variations might be suppress the oxygenating activity of the enzyme[14]
more effective than the distant ones in perturbing This residue is a strictly conserved and essential for
enzyme enantioselectivity. Table 1 provides an inven- activity for the other Baeyer–Villiger monooxygenas-
tory of the amino acids with a theoretical pKa close to es.[15] As already reported, Arg337 is located inside
the values determined from the titration curves the active site, on the re side of the flavin.[8] Judging
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Francesca Zambianchi et al.
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detector) equipped with a Chiracel OD (Daicel) chiral
column. Chiral GC analysis: Hewlett–Packard 6890 Series
gas chromatograph equipped with a CP-Chirasil-DEX-CB
chiral column.
from the crystal structure, Arg337 appears to be able
to directly interact with the Criegee intermediate.[8]
Furthermore, Arg337 can adopt two alternate confor-
mations, which underlines an inherent flexibility that
can be functionally important.[8]
Enzymatic Oxidation of Thioanisole
The pH dependence of PAMO stereoselectivity
seems to point i) to the equilibrium FAD:C4a-hydro-
The reactions were carried out at 278C in 1 mL of 50 mM
peroxide/FAD:C4a-peroxide and ii) to the protona- Tris/HCl buffer, pH 6–10, containing 1 mg of thioanisole,
1 mg of glucose 6-phosphate, 0.5 U of PAMO and 10 U of
glucose 6-phosphate dehydrogenase. The mixture was
shaken at 250 rpm in a rotatory shaker for the times estab-
lished. The reactions were then stopped, worked up by ex-
traction with dichloromethane (3”0.5 mL), dried over
Na2SO4 and analyzed by chiral HPLC[6b] in order to deter-
mine the conversion and the enantiomeric excesses of the
sulfoxide. Standard deviations (average ꢃ 7 measurements)
were below ꢀ1.5%. The pKa value was obtained by non-
linear least-square fit of values according to the Henderson–
tion state of Arg337 as the two crucial factors for
fine-tuning of enzyme stereochemical outcome. This
hypothesis is supported by the fact that PAMO carries
out the S-oxidation and the Baeyer–Villiger reaction
with two distinct mechanisms, that is, electrophilic
attack to the heteroatom (S-oxidation) and nucleo-
philic attack to the carbonyl group (Baeyer–Villiger
reaction). The electrophilic attack to electron-rich
substrates is the typical reaction of another flavopro-
tein monooxygenase: 4-hydroxybenzoate 3-monooxy- Hasselbach equation.
genase, where the protonation of the distal oxygen of
the peroxide moiety increases the electrophilic reac- Enzymatic Oxidation of 2-Phenylpropionaldehyde
tivity.[16] In S-oxidation of dimethyl sulfide by lumifla-
vin, theoretical studies indicated that the lowest acti-
vation energy of the transition state was obtained
pionaldehyde, 1 mg of glucose 6-phosphate, 0.5 U of PAMO
The reactions were carried out at 278C in 1 mL of 50 mM
Tris/HCl buffer, pH 6–10, containing 1 mg of 2-phenylpro-
with the complex formed with FAD:C4a-hydroperox-
ide-water, which suggests that the electrophilic attack
is preferred over the nucleophilic one.[17] We think
that among FAD:C4a-hydroperoxide, protonated
Arg337 and aqueous buffer, FAD:C4a-hydroperoxide
mostly influenced the geometry of electrophilic attack
to the sulfur atom, and therefore, the stereochemical
arrangement of the transition state. Instead, in the
Baeyer–Villiger oxidation of 2-phenylpropionalde-
hyde the stereochemical arrangement of the transition
state is mainly influenced by the presence of proton-
ated Arg337.
and 10 U of glucose-6-phosphate dehydrogenase. The mix-
ture was shaken at 250 rpm in a rotatory shaker for the
times established. The reactions were then stopped, worked
up by extraction with dichloromethane (3”0.5 mL), dried
over Na2SO4. The ee values of the product, from which the
enantiomeric ratios were derived,[10] were determined by
chiral GC.[18] Standard deviations (average ꢃ 5 measure-
ments) were below ꢀ1.5. The pKa value was obtained by
non-linear least-square fit of values according to the Hen-
derson–Hasselbach equation.
In conclusion, this study shows for the first time
that is possible to take advantage of the enantioselec-
tive properties of an enzyme to titrate and assign
some residues important for catalysis. The protona-
tion of Arg337 and the FAD:C4a-hydroperoxide/
FAD:C4a-peroxide equilibrium were identified as the
two major factors responsible for the fine-tuning of
PAMO enantioselectivity.
Acknowledgements
CERC3 and COST D25/0005/03 are gratefully acknowl-
edged for funding and support.
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Recombinant histidine-tagged phenylacetone monooxyge-
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