Communications
Table 2: Catalytic activity and enantioselectivity for ethylbenzene hydrox-
ylation by P450BSb in the presence of carboxylic acids.
catalyzes the hydroxylation reaction of the natural substrate
[
28,29]
[30]
(fatty acid)
and the transformation of indole to indigo.
By shortening the alkyl-chain length of the natural
substrates, we have demonstrated a novel approach for the
introduction of new functions and higher activities into
P450BSb while keeping its intrinsic advantage, namely, the
use of H O . By designing the nature and structure of the
decoy molecule, P450BSb could be tailored to accommodate
and oxidize a wide range of substrates. A combination of the
decoy molecule with mutagenesis would enable us to create
P450BSb systems that catalyze various hydroxylation reactions
without consuming expensive cofactors, such as NAD(P)H.
À1 [a]
Carboxylic acid
Rate [min
]
ee (R) [%]
2
2
butanoic acid (C4)
pentanoic acid (C5)
hexanoic acid (C6)
heptanoic acid (C7)
octanoic acid (C8)
11Æ1
20Æ2
24Æ2
28Æ4
10Æ3
35Æ4
41Æ4
51Æ3
68Æ2
61Æ3
À1
À1
[
a] The unit for catalytic activity is (nmol product)min (nmol P450)
;
uncertainty given as the standard deviation for five measurements.
Received: January 6, 2007
Published online: March 27, 2007
Keywords: cytochrome P450 · enzyme catalysis ·
.
À1
hydrogen peroxide · hydroxylation
a rate of 28 turnovermin (68% ee) in the presence of
heptanoic acid (Table 2). These results clearly indicate that
P450BSb misrecognizes short-alkyl-chain carboxylic acids as
the substrate owing to its structural similarity and catalyzes
the oxidation of non-fatty acids. The rate of oxidation of
[
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is reported in the range 159–
whereas that of the guaiacol is
0fold higher. The results imply that the decoy molecule
BSb
1
À1 [20,24]
3
65 turnovermin ,
[
1
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keeps the P450BSb catalytic cycle always “on”, whereas the
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oxidation cycle “on”. Furthermore, myristic acid was found to
prohibit the nonnatural substrate oxidation, possibly because
of the full occupation of the space of the heme vicinity by the
alky chain.
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[
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The high enantioselectivity observed for the styrene
epoxidation and the ethylbenzene hydroxylation suggests
that the specific substrate binding site could be constructed by
the combination of the protein scaffold and the decoy
molecule. As the enantioselectivity of the ethylbenzene
hydroxylation is extremely dependent on the structure of
the decoy molecules, higher selectivity can be introduced by
using chiral carboxylic acids. The initial turnover rates for
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BSb
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2
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greater than those of chloroperoxidase (CPO), which is one
of the most efficient hydrogen peroxide dependent biocata-
lysts. For example, wild-type CPO catalyzes the hydroxylation
of ethylbenzene and the epoxidation of styrene at rates of 1.4
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[
À1
À1
and 288 (nmol product)min (nmol CPO) , respec-
tively.
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26,27]
That the catalytic activities are dependent on the
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alkyl-chain structure of the decoy molecule indicates that the
catalytic activities may be further improved by the design of
the decoy molecule.
[
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As described herein, we have observed versatile H O -
2
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dependent monooxygenase activities including the hydroxyl-
ation of ethylbenzene into P450BSb without replacing any
amino acid residues. To the best of our knowledge, this is the
first example of the drastic change of substrate specificity
without any mutagenesis. Furthermore, the efficient H O -
[
[
2
2
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658
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2007, 46, 3656 –3659