S-sulfoxide in 47% ee, even in the complete absence of
vanadium.
with the above proposed formation of peroxycarboxylic acids.
Appropriate site-directed mutagenesis studies should be able to
confirm the key role of Asp339 in the catalytic mechanism.
In conclusion, we have demonstrated the feasibility of
rationally designing a semi-synthetic peroxidase via incorpora-
tion of vanadium into the active site of phytase by exploiting the
structural similarity of vanadate to phosphate. Surprisingly, the
metal-free phytase also catalyses enantioselective oxidation and
further studies are underway aimed at clarifying the mechanism
and exploring the scope of this novel oxygen transfer catalysed
by a hydrolase.
We subsequently studied the metal-free phytase-catalysed
sulfoxidation in more detail (Table 1). The conversion and ee
increased with increasing enzyme concentration, consistent
with the reaction being enzyme catalysed. At room temperature
the enzyme activity for the two enantiomers was 0.16 µmol h21
(mg protein)21 and 0.04 µmol h21 (mg protein)21 respectively
for the S- and the R-enantiomer. In consequence, the intrinsic
enantioselectivity of the enzyme is 58% ee (S) at room
temperature. In the absence of enzyme, a 34% conversion to
racemic sulfoxide was observed in 5 h at 40 °C. Decreasing the
temperature from 40 to 4 °C reduced the rate of the blank
We gratefully acknowledge a gift of phytase by Gist-
brocades N.V. The financial support of the Dutch Innovation
Oriented Program on Catalysis (IOP catalysis; IKA94013) is
gratefully acknowledged.
reaction from 9.52 1028 to 3.42 1029
M
s21, resulting in a
corresponding increase in enantioselectivity from 33 to 54%
ee.
The highest ees were observed in formate buffer, but a
carboxylate buffer was not essential. Ammonium chloride and
MES‡ were also effective, giving conversions and (ee values)
after 5 h of 74% (35%) and 71% (43%), respectively, in
Notes and References
† E-mail: Secretariat-OCK@stm.tudelft.nl
‡ 2-(N-Morpholino)ethanesulfonic acid
§ 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
experiments performed at 4 m H2O2. This rules out the in situ
M
formation of peroxycarboxylic acids (from the carboxylate
buffers) as observed with lipases11 and metal-free bacterial
haloperoxidases.12 The optimum pH was 5.1 which coincides
with that of the natural reaction of phytase. The enantioselectiv-
ity decreased from 33 to 18% with increasing H2O2 concentra-
tion from 5.5 to 20 m , owing to an increased contribution from
the blank reaction at higher H2O2 concentrations.
A few experiments were performed aimed at providing
insights into the origins of the observed metal-free catalysis.
Reactions performed under nitrogen showed no difference with
the reactions performed under air. When the reaction was
performed with H218O2 (Campro Scientific) analysis of the
sulfoxide product by GC–MS showed that the oxygen (100%) is
derived from H2O2. Addition of the radical scavenger Tro-
loxTM-C,§ a water soluble analog of vitamin E, had no influence
on the conversion or enantioselectivity, consistent with hy-
droxyl radicals not being involved in the enantioselective
oxidation.
1 M. P. J. van Deurzen, F. van Rantwijk and R. A. Sheldon, Tetrahedron,
1997, 53, 13 183.
2 S. Colonna, N. Gaggero, G. Carrea and P. Pasta, Chem. Commun., 1997,
439; S. Colonna, N. Gaggero, L. Casella, G. Carrea and P. Pasta,
Tetrahedron: Asymmetry, 1992, 3, 95; M. P. J. van Deurzen, I. J.
Remkes, F. van Rantwijk and R. A. Sheldon, J. Mol. Catal. A: Chem.,
1997, 117, 329.
M
3 M. P. J. van Deurzen, K. Seelbach, F. van Rantwijk, U. Kragl and R. A.
Sheldon, Biocatal. Biotrans., 1997, 15, 1.
4 J. W. P. M. van Schijndel, E. G. M. Vollenbroek and R. Wever, Biochim.
Biophys. Acta, 1993, 1161, 249; J. W. P. M. van Schijndel, P. Barnett,
J. Roelse, E. G. M. Vollenbroek and R. Wever, Eur. J. Biochem., 1994,
225, 151; A. Messerschmidt and R. Wever, Proc. Natl. Acad. Sci.
U.S.A., 1996, 93, 392.
5 M. Andersson, A. Willetts and S. Allenmark, J. Org. Chem., 1997, 62,
8455.
6 W. Hemrika, R. Renirie, H. L. Dekker, P. Barnett and R. Wever, Proc.
Natl. Acad. Sci. U.S.A., 1997, 94, 2145; A. F. Neuwald, Protein Sci.,
1997, 6, 1764.
This leaves us with the question of the origin of the observed
oxygen transfer catalysis. It is not metal-based as there is no
metal ion in the active site. Although the chelating agent EDTA
completely inhibited the enantioselective oxidation this is
probably due to removal of calcium ions required for the
stability of the enzyme. The crystal structure of phytase from
Aspergillus ficuum was recently resolved to 2.5 Å, showing that
an aspartate residue (Asp339) is located in the active site.13
Similarly, acid phosphatases are also known to contain aspartate
in the active site.7 Hence, it is tempting to speculate that this
aspartate plays a key role in the observed catalysis. Reaction of
the free carboxylate group with H2O2 would give the corre-
sponding peroxycarboxylic acid which could be the active
oxidant. When the reaction was performed with tert-butyl
hydroperoxide (TBHP) no catalysis was observed consistent
7 Y. Lindqvist, G. Schneider and P. Vihko, Eur. J. Biochem., 1994, 221,
139; C. M. Vescina, V. C. Salice, A. M. Cortizo and S. B. Etcheverry,
Biol. Trace Elem. Res., 1996, 53, 185.
8 A. Mahajan and S. Dua, J. Agric. Food Chem., 1997, 45, 2504.
9 P. J. Stankiewicz and M. J. Gresser, Biochemistry, 1988, 27, 206.
10 M. T. Pope, Heteropoly and Isopoly Oxometalates, Springer-Verlag,
Berlin, 1983, p. 34.
11 M. C. de Zoete, F. van Rantwijk, L. Maat and R. A. Sheldon, Recl. Trav.
Chim. Pays-Bas, 1993, 112, 462.
12 M. Picard, J. Gross, E. Lübbert, S. Tölzer, S. Krauss, K.-H. van Pée and
A. Berkessel, Angew. Chem., Int. Ed. Engl., 1997, 36, 1196.
13 D. Kostrewa, F. Grüninger-Leitch, A. D’Arcy, C. Broger, D. Mitchell
and A. P. G. M. van Loon, Nat. Struct. Biol., 1997, 4, 185.
Received in Cambridge, UK, 22nd June 1998; 8/04702B
1892
Chem. Commun., 1998