Direct hydroxylation of primary carbons in small alkanes by wild-type cytochrome P450BM3 containing perfluorocarboxylic acids as decoy molecules

Article abstract of DOI:10.1039/c3sc50378j

Cytochrome P450BM3 (P450BM3) is a long-alkyl-chain fatty acid hydroxylase that shows an extremely high catalytic turnover rate and high coupling efficiency of NADPH for product formation (product formation rate per NADPH consumption rate). Although P450BM3 exclusively hydroxylates long-alkyl-chain fatty acids, we have found that simple addition of perfluorocarboxylic acids (PFs) as inert dummy substrates (decoy molecules) turns P450BM3 into a small alkane hydroxylase. For example, PF-bound P450BM3 oxidizes propane and butane to 2-propanol and 2-butanol, respectively. The coupling efficiency of small alkane hydroxylation, however, is very low compared with that of long-alkyl-chain fatty acid hydroxylation. In this study, we examined the experimental conditions for small alkane hydroxylation in an effort to improve the coupling efficiency and to realize the hydroxylation of their primary carbons. To increase the concentration of gaseous substrates in the reaction mixture, we performed reactions under the high-pressure condition of 0.5 MPa small alkanes. Propane hydroxylation under high-pressure conditions significantly improved the coupling efficiency to 48%. Furthermore, 1-propanol, which has never been observed under lower-pressure conditions, was produced. It is noteworthy that a detectable amount of ethanol was observed in the ethane hydroxylation under the pressure condition of 0.5 MPa, whereas methane was not hydroxylated. These results indicate that by increasing the concentration of small alkanes, the "P450BM3-decoy molecule system" can catalyze hydroxylation reactions of the primary carbons in small alkanes.

Full text of DOI:10.1039/c3sc50378j

Showcasing research from Prof. Yoshihito Watanabe’s
laboratory, Nagoya University, Nagoya, Japan.
As featured in:
Direct hydroxylation of primary carbons in small alkanes by wild-
type cytochrome P450BM3 containing perfluorocarboxylic acids
as decoy molecules
Simple addition of perfluorocarboxylic acids as inert dummy
substrates (decoy molecules) turns cytochrome P450BM3
(P450BM3) into a small alkane hydroxylase, although P450BM3
exclusively hydroxylates long-alkyl-chain fatty acids. Hydroxylation
of the primary carbons of propane and ethane was catalyzed by
a “P450BM3–decoy molecule system”by performing the reaction
under a high pressure of gaseous alkane (0.5 MPa).
See N. Kawakami, O. Shoji
and Y. Watanabe,
Chem. Sci., 2013, 4, 2344.
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Direct hydroxylation of primary carbons in small alkanes
by wild-type cytochrome P450BM3 containing
perfluorocarboxylic acids as decoy molecules†
Cite this: Chem. Sci., 2013, 4, 2344
a
b
a
Norifumi Kawakami, Osami Shoji* and Yoshihito Watanabe*
Cytochrome P450BM3 (P450BM3) is a long-alkyl-chain fatty acid hydroxylase that shows an extremely high
catalytic turnover rate and high coupling efficiency of NADPH for product formation (product formation
rate per NADPH consumption rate). Although P450BM3 exclusively hydroxylates long-alkyl-chain fatty
acids, we have found that simple addition of perfluorocarboxylic acids (PFs) as inert dummy substrates
(
decoy molecules) turns P450BM3 into a small alkane hydroxylase. For example, PF-bound P450BM3
oxidizes propane and butane to 2-propanol and 2-butanol, respectively. The coupling efficiency of small
alkane hydroxylation, however, is very low compared with that of long-alkyl-chain fatty acid
hydroxylation. In this study, we examined the experimental conditions for small alkane hydroxylation in
an effort to improve the coupling efficiency and to realize the hydroxylation of their primary carbons. To
increase the concentration of gaseous substrates in the reaction mixture, we performed reactions under
the high-pressure condition of 0.5 MPa small alkanes. Propane hydroxylation under high-pressure
conditions significantly improved the coupling efficiency to 48%. Furthermore, 1-propanol, which has
never been observed under lower-pressure conditions, was produced. It is noteworthy that a detectable
amount of ethanol was observed in the ethane hydroxylation under the pressure condition of 0.5 MPa,
whereas methane was not hydroxylated. These results indicate that by increasing the concentration of
small alkanes, the “P450BM3–decoy molecule system” can catalyze hydroxylation reactions of the
primary carbons in small alkanes.
Received 8th February 2013
Accepted 15th March 2013
DOI: 10.1039/c3sc50378j
www.rsc.org/chemicalscience
number of P450BM3 mutants that oxidize a variety of non-
natural substrates, such as small alkanes and molecules having
Introduction
Cytochrome P450BM3 is a P450 family enzyme catalyzing the steroidal backbones, have been successfully prepared.12,13
hydroxylation of subterminal carbons of long-alkyl-chain fatty Although crystal structures of P450BM3 mutants containing
1,2
acids (Fig. 1). Unlike most P450s, P450BM3 is a catalytically non-natural substrates are not available, these studies suggest
self-sufficient P450 whose reductase domain is fused with the that reshaped active sites of P450BM3 for binding of non-
1,3,4
12,13
P450 domain on the same peptide chain.
This efficient
natural substrates were constructed by mutagenesis.
activation system confers on P450BM3 an extremely high cata-
Appropriate binding of a substrate to the active site of P450BM3
is also important for initiating the generation of active species,
À1
lytic activity with a turnover number of up to 17 000 min
,
which is the highest oxidase activity among the P450s reported namely an oxoferryl(IV) porphyrin p cation radical (so-called
5
14–16
thus far. In addition to these properties, a practical amount of compound I).
the enzyme can be obtained as a soluble form by using a typical fatty acid binding, which results in the removal of a water
The catalytic cycle of P450BM3 involves (1)
1,6
Escherichia coli gene expression system. Thus, P450BM3 has molecule ligated to the heme iron to cause the positive redox
been extensively studied as a potent biocatalyst, particularly potential shi of the heme iron, (2) reduction of Fe to Fe by
3
+
2+
using protein engineering to produce enzymes having desired
the rst electron transfer from NAD(P)H through the reductase
domain, (3) binding of molecular oxygen to the ferrous heme,
7–11
17
activities based on the mutagenesis technique.
In fact, a
(
4) production of the active oxidant species (compound I) by
a
Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, reductive activation of the molecular oxygen through the second
Nagoya, 464-8602, Japan. E-mail: p47297a@nucc.cc.nagoya-u.ac.jp; Fax: +81-52-
electron transfer from NAD(P)H, and (5) oxidation of the bound
7
89-2953; Tel: +81-52-789-2953
18–21
fatty acid by compound I.
According to the reaction mech-
b
Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho,
Chikusa-ku, Nagoya, 464-8602, Japan. E-mail: shoji.osami@a.mbox.nagoya-u.ac.jp;
Tel: +81-52-789-3557
anism of P450BM3, a molecule having a structure very different
from that of a fatty acid cannot initiate the rst step of the
catalytic cycle. Conversely, if the size and shape of the active site
of P450BM3 are reshaped by the addition of a small molecule
†
Electronic supplementary information (ESI) available: GC/MS analysis. See DOI:
0.1039/c3sc50378j
1
2
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Fig. 1 Reaction mechanisms of the natural reaction system (left) and the decoy molecule system (right) of P450BM3. The subterminal carbons of fatty acids are
hydroxylated in the natural reaction (left pathway). By simple addition of a decoy molecule such as PFC10, the hydroxylation reaction of a small alkane (for example,
ethane) is catalyzed by P450BM3 because of substrate misrecognition of P450BM3 (right pathway). The active site crystal structure of P450BM3 containing natural
substrate (PDB code: 1FAG) and a plausible structure of P450BM3 containing PFC10 and ethane are shown in the respective reaction pathways.
that can bind to the active site, even wild-type P450BM3 would presence of PFs, compound I is further reduced back to the
25
provide
a
binding pocket to accommodate non-natural resting state, with the production of a water molecule. Because
this uncoupling reaction and the small alkane hydroxylation
substrates and could oxidize them without any mutagenesis.
Based on these considerations, recently, our research group reaction compete with each other, the coupling efficiency can be
and Zilly et al. independently developed a novel method to alter improved by increasing the concentration of small alkane to
the substrate specicity of wild-type P450BM3 from long-alkyl- compensate for its low binding affinity to the active site of a
chain fatty acids to small gaseous alkanes by simple addition of P450BM3–PF complex.
22,23
inert dummy substrates (decoy molecules) (Fig. 1).
The
We herein report the effect of the small alkane concentra-
decoy molecules are a series of peruorocarboxylic acids (PFs) tion, investigated by pressurizing the reaction vessel with
with alkyl chains containing 8–14 carbon atoms (PFC8–PFC14). 0.5 MPa (5 bar) of small alkane using an autoclave, on both the
It was thought that the PFs would bind to the active site of coupling efficiency and the catalytic activity (Fig. 2).
P450BM3 through interactions of their carboxylic group with
Arg47 and Tyr51, similar to the natural substrate binding
manner, but PFs are never oxidized by P450BM3 because of
Experimental methods
Materials
their inert C–F bonds (C–F bond dissociation energy: 116 kcal
mol ). We found that PFC9 and PFC10, having shorter alkyl
À1
P450BM3 was prepared according to a method previously
described. Methane (99.999% min) was purchased from Japan
Fine Products. Ethane (99.99% min), propane (99.5% min), and
a gas mixture containing 7% methane, 4% oxygen, and 89%
nitrogen were purchased from Takachiho Chemical Industrial
Co. Butane (99% min) and nitrogen gas were purchased from
chains than those of the natural substrates (C12–20), are
effective for propane and butane hydroxylation reactions. We
therefore concluded that the PFs would partially occupy the
active site of P450BM3 and provide space, of suitable size and
22
24
shape, to accommodate these small alkanes (Fig. 1). The
P450BM3–decoy molecule system” also catalyzes the hydroxy-
lation of octane, hexane, cyclohexane, and branched
“
22,23
alkanes.
Although small alkanes were converted to the corresponding
alcohols by wild-type P450BM3 in the presence of PFs, the
coupling efficiencies (product formation rate per NADPH
consumption rate) were very low: 18% and 55% for propane and
butane hydroxylation reactions, respectively. A possible reason
for the low coupling efficiencies is the low binding affinity of
small alkanes, leading to an uncoupling reaction. According to
Fig. 2 Schematic of the pressurizing system. The reaction vessel is pressurized
a report regarding the uncoupling reaction of P450BM3 in the with 0.5–1.0 MPa small alkane using an autoclave.
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Sumitomo Seika Chemicals Co. and Iwatani Corporation,
respectively. PFC8 and PFC9 were purchased from Fluorochem
Results and discussion
and TCI Co., respectively. PFC10 and glucose dehydrogenase We rst carried out the hydroxylation reaction of propane by
GDH) were purchased from Wako Pure Chemical Industries. P450BM3 under a high-pressure condition of 0.5 MPa propane
(
NADPH and glucose were purchased from Nacalai Tesque. gas in the presence of PFC10. By pressurizing the reaction
+
NADP was purchased from Oriental Yeast Co.
vessel, the amount of 2-propanol was increased from 700 mM to
2
4
170 mM and the coupling efficiency was improved from 18% to
8% (Table 1). Furthermore, a 1-propanol peak, which has
22
Propane hydroxylation under high pressure of propane
never been observed in previous reports, was observed by GC
analysis (Fig. 3a).
5
A buffer solution (20 mM Tris–HCl: pH 7.4) containing 5 mM
NADPH and 100 mM PFC10 was placed in an autoclave with a
magnetic stirrer bar that had a small depression on the top. A
drop of P450BM3 solution was placed in the small depression of
the stirrer bar, to separate it from the reaction mixture (Fig. 2).
The autoclave was placed in a water bath and pressurized with
small alkane to 0.5 MPa, without stirring. Aer 1 min incuba-
tion, the reaction was initiated by stirring the stirrer bar,
thereby mixing the drop of P450BM3 with the reaction solution
22,23,27
The concentration of 1-propanol was
0 mM (Table 1). In contrast, the control reaction, in which inert
nitrogen gas (0.5 MPa) was used instead of propane gas, gave a
peak of negligible intensity at the retention time corresponding
to 1-propanol (Fig. 3a), indicating that the primary carbon of
small alkanes can be oxidized by wild-type P450BM3.
With this progress in mind, the oxidation of the primary
carbons of ethane was examined under the pressure condition
of 0.5 MPa ethane using PFC10 as a decoy molecule: 220 mM of
ethanol was formed within 2 h (Fig. 3b). To estimate the amount
of background ethanol, a control experiment using inert
nitrogen gas (0.5 MPa) instead of ethane gas was also carried
out. Even in the absence of ethane gas, 140 mM ethanol was
detected. Aer deducting the background ethanol, up to 80 mM
(nal concentration of P450BM3 was 500 nM). The reaction was
carried out for 1 h, aer which the remaining amount of NADPH
was measured by absorbance at 340 nm. The product alcohol
was derivatized to a nitrite ester according to a published
22,26
method.
by gas chromatography (GC/FID) with an Rtx-1 column (Restek:
0 m, 0.53 mm ID, 3 mm D ) and by GC/MS with a TC-Bond Q
The concentrations of the derivatives were analyzed
À1
À1
(40 nmol h nmol -P450BM3) of ethanol was assignable to
6
f
the product of ethane gas hydroxylation. This result indicates
that even the primary carbon of ethane can be hydroxylated by a
column (GL Sciences: 30 m, 0.32 mm ID, 10 mm Df).
“P450BM3–decoy molecule system” when the concentration of
Ethane hydroxylation under a pressure condition of 0.5 MPa
ethane in the reaction mixture is increased under a high pres-
sure of ethane, whereas turnover frequency is very low. In
general, detection of a small amount of ethanol is very difficult
because of the contamination of ethanol from reagents used in
the oxidation. We carefully estimated the amount of ethanol
contamination from the reagents to be 60 mM and noticed that
the amount of ethanol observed in the control experiment
Ethane hydroxylation under the pressure of 0.5 MPa ethane was
performed in the same manner as the propane hydroxylation
except that the concentrations of P450BM3 and NADPH were
increased to 1 mM and 5–15 mM, respectively. Reactions were
carried out for 1–3 h. The amounts of ethanol and NADPH
consumed were determined by GC and UV-vis spectroscopy,
respectively. To determine the background ethanol formation,
control reactions under a pressure condition of 0.5 MPa
nitrogen gas instead of ethane or methane were performed. The
effect of PFC10 was also examined by comparing the reactions
in the absence and presence of PFC10. The peak intensities
of the produced alcohols were normalized to the intensity of
(without ethane) was larger than the contamination. Appar-
ently, the increase in background ethanol during the reaction is
attributable to a side reaction, other than ethane hydroxylation;
we observed that the increase in background ethanol was
28
proportional to the amount of NADPH consumption (Fig. 4).
Finally, we attempted the hydroxylation of methane gas
under the pressure condition of 0.5 MPa methane. Although we
observed a peak attributable to methanol with a relatively
strong intensity (corresponding to 2.9 mM methanol) in GC
analysis, a control experiment using 100% nitrogen gas also
showed the same peak at the same retention time and with the
3-pentanol (internal standard).
Methane hydroxylation under a pressure condition of 0.5 or
.0 MPa
1
Methane hydroxylation under the pressure condition of 0.5 MPa same area intensity, suggesting that the methanol observed was
29,30
was performed in the same manner as described for the ethane not the product of methane hydroxylation (Fig. 3c).
hydroxylation. In addition, to assess the reproducibility of the
According to a report by Zilly et al., however, methane was
23
methane hydroxylation reaction reported by Zilly et al., we also very efficiently converted to methanol by the same “P450BM3–
performed the reaction under a pressure condition of 1.0 MPa decoy molecule system” and a 2472 turnover number (TON) was
23
using a gas mixture containing 7% (70 kPa) methane, 4% reported for the methane hydroxylation. We therefore per-
(
40 kPa) oxygen, and 89% (890 kPa) nitrogen with 1 mM PFC10 formed the methane hydroxylation under essentially the same
and a NADPH regeneration system composed of 1 mM NADP , conditions as those reported by Zilly et al.: (1) 1.0 MPa (10 bar)
+
1
unit GDH, and 100 mM glucose, instead of the addition of gas mixture containing 7% methane (partial pressure of
NADPH. The reaction was carried out for at least 17.5 h. methane 70 kPa), 4% oxygen (40 kPa), and 89% nitrogen
Methanol was detected by GC/FID. The peak intensity of the (890 kPa), (2) 1 mM PFC10 as a decoy molecule, and (3) a
methanol was normalized to the intensity of 3-pentanol.
NADPH regeneration system composed of 100 mM glucose,
2
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ꢀ
Table 1 Catalytic activity of propane hydroxylation by P450BM3 (500 nM) with PFC10 (100 mM) at 20 C under the pressure condition of 0.5 MPa propane. The
amounts of 2-propanol and 1-propanol are given in mM. The amount of NADPH consumed and the coupling efficiency are given in mM and as [product]/[NADPH
consumed] Â 100, respectively
a,b
a,b
a,b
a,c
2
-Propanol /mM
1-Propanol /mM
NADPH consumption /mM
Coupling efficiency /%
2
170 Æ 30 (4340)
50 Æ 2 (100)
4490 Æ 30 (8980)
48 Æ 1(50)
a
b
À1
Values are averages Æ standard deviations calculated from three different experiments. Values in parentheses are turnover frequency (nmol h
À1
c
nmol -P450BM3). Value in parentheses calculated based on the amounts of 1-propanol and 2-propanol.
Fig. 4 NADPH-dependent ethanol formation. The ethanol produced from the
reaction under 0.5 MPa (absolute pressure 0.6 MPa, containing 3.3% O , 13.3%
, and 83.3% substrate) of ethane (C), nitrogen (B), and methane (,), and in
2
N
2
the absence of PFC10 with ethane (O), and contamination of ethanol from the
reagents (:) are plotted against the amount of NADPH consumption. The dotted
2
line in the figure is the line fitted based on control experiments with N , methane,
Fig. 3 GC analysis of the hydroxylation of propane (a), ethane (b), and methane
c) by P450BM3 with PFC10 under the pressure condition of 0.5 MPa. Panel (d)
shows GC charts of the hydroxylation of methane using a gas mixture (7% CH
% O , and 89% N ) under the pressure condition of 1.0 MPa (see main text for
and contamination ethanol, showing the background ethanol. This figure clearly
shows that the concentration of the background ethanol is proportional to the
amount of NADPH consumed. Furthermore, ethanol was produced even when
methane gas was used instead of ethane gas. Thus the background ethanol was
produced from the uncoupling reactions induced by the PFC10. In contrast, the
concentrations of ethanol produced in the presence of ethane gas were signifi-
cantly higher than the estimated background ethanol (dotted line) and the
differences from the background ethanol also increased as the NADPH
consumption increased.
(
4
,
4
2
2
details). Insets in panels (a) and (d) are magnified profiles of the 1-propanol and
methanol, respectively. Blue lines show the peaks of 2-propanol (a) 5.18 min, 1-
propanol (a) inset 5.89 min, ethanol (b) 4.21 min, and methanol (c) 3.43–3.5 min,
obtained from the reaction in the presence of propane, ethane, and methane,
respectively. The orange lines represent control experiments, to estimate the
background ethanol and methanol under conditions without small alkanes.
Nitrogen (0.5 MPa) was used in the control experiment reactions instead of small
alkanes. The peak intensities of the alcohols were normalized to the intensity of 3-
pentanol (internal standard).
À1
energy of methane (104.9 kcal mol ), the turnover frequency
for the methane hydroxylation under the conditions examined
here would be too low to detect by GC, even if methane is
hydroxylated. In fact, in a recent report on methane hydroxyl-
ation by CYP153A6 using iodosylbenzene as an oxidant, a large
excess amount of P450 (50–250 mM) was used to detect a small
amount of methanol (0.02–0.05 TON) that originated from
+
1
mM NADP , and 1 unit GDH instead of the addition of
NADPH. First, we carried out a control experiment using 100%
nitrogen gas (1.0 MPa) to estimate the background methanol
and observed ca. 50 mM methanol. Compared with the reaction
system utilizing NADPH, the background methanol was dras-
tically decreased in the case where the NADPH regeneration
system was used. However, the area intensity of methanol was
not increased (Fig. 3d) even when the reaction was performed
32
methane gas hydroxylation.
Conclusions
using a gas mixture containing methane as a substrate. This Hydroxylation of the primary carbons of propane and ethane
indicated that the methanol observed in our experiment did not was catalyzed by a “P450BM3–decoy molecule system” by per-
31
originate from methane hydroxylation.
The turnover forming the reaction under a high pressure of gaseous alkane
frequencies for the formation of 2-propanol, 1-propanol, and (0.5 MPa), which effectively increases the concentration of the
ethanol by the P450BM3–PFC10 system were estimated to be small alkane in the reaction solution. The concentration of the
À1
4
340, 100, and 40 h , respectively. These values are a good small alkane governs the efficiency of the reaction by a
reection of the C–H bond dissociation energy of the secondary “P450BM3–decoy molecule system”.
À1
carbon of propane (98.6 kcal mol ), the primary carbon of
Thus we obtained 1-propanol and ethanol despite the barrier
À1
propane (100.4 kcal mol ), and the primary carbon of ethane of high C–H bond dissociation energies of primary carbons, but
101.1 kcal mol ). Considering the C–H bond dissociation methane hydroxylation is still difficult to produce a detectable
À1
(
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amount of methanol because of its large C–H bond dissociation 16 S. R. Bell and J. T. Groves, J. Am. Chem. Soc., 2009, 131, 9640–
À1
energy, 104.9 kcal mol . We believe that our reaction system
9641.
could be further improved by designing a decoy molecule 17 R. Perera, M. Sono, G. M. Raner and J. H. Dawson, Biochem.
leading to both high activity and high coupling efficiency for
Biophys. Res. Commun., 2005, 338, 365–371.
ethane hydroxylation.
18 S. G. Sligar, Biochemistry, 1976, 15, 5399–5406.
19 S. N. Daff, S. K. Chapman, K. L. Turner, R. A. Holt,
S. Govindaraj, T. L. Poulos and A. W. Munro, Biochemistry,
Acknowledgements
1997, 36, 13816–13823.
A plasmid encoding P450BM3 was kindly supplied by Professor 20 M. B. Murataliev and R. Feyereisen, Biochemistry, 1996, 35,
S. G. Sligar, University of Illinois, USA. This work was supported
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by Grants-in-Aid for Scientic Research (S) to Y. W. (24225004) 21 T. W. Ost, J. Clark, C. G. Mowat, C. S. Miles,
from the Ministry of Education, Culture, Sports, Science, and
Technology (Japan) and a Grant-in-Aid for Young Scientists (A)
to O. S. (21685018).
M. D. Walkinshaw, G. A. Reid, S. K. Chapman and S. Daff,
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22 N. Kawakami, O. Shoji and Y. Watanabe, Angew. Chem., Int.
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2
3 F. E. Zilly, J. P. Acevedo, W. Augustyniak, A. Deege,
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Notes and references
1
2
L. P. Wen and A. J. Fulco, J. Biol. Chem., 1987, 262, 6676–6682.
S. S. Boddupalli, B. C. Pramanik, C. A. Slaughter, 24 The PFs binding at the entrance of the active site of
R. W. Estabrook and J. A. Peterson, Arch. Biochem. Biophys.,
992, 292, 20–28.
P450BM3 would also assist the connement of small
alkane substrates.
1
3
4
L. O. NarhiandA. J. Fulco, J. Biol. Chem., 1987, 262, 6683–6690. 25 N. Pons, S. Pipino and F. De Matteis, Biochem. Pharmacol.,
J. A. Peterson and S. S. Boddupalli, Arch. Biochem. Biophys.,
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T. W. Ost, C. S. Miles, J. Murdoch, Y. Cheung, G. A. Reid,
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26 H. T. Nguyen, N. Takenaka, H. Bandow and Y. Maeda, Anal.
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1
5
6
7
S. K. Chapman and A. W. Munro, FEBS Lett., 2000, 486, 27 1-Propanol was also detected by GC/MS analysis (see
73–177.
Fig. S1†).
1
C. J. C. Whitehouse, S. G. Bell, W. Yang, J. A. Yorke, 28 The average turnover frequency with standard deviation of
À1
C. F. Blanford, A. J. F. Strong, E. J. Morse, M. Bartlam,
Z. Rao and L.-L. Wong, ChemBioChem, 2009, 10, 1654–1656.
H. M. Girvan, A. J. Dunford, R. Neeli, I. S. Ekanem,
T. N. Waltham, M. G. Joyce, D. Leys, R. A. Curtis,
ethanol formation was calculated to be 40 Æ 10 h based
on data points obtained from 2 and 3 h reactions (C
plotted against NADPH consumption more than 8.9 mM in
Fig. 4).
P. Williams and K. Fisher, Arch. Biochem. Biophys., 2011, 29 We observed ethanol formation even in the methane
5
07, 75–85.
hydroxylation reaction (Fig. 4 ,). The amount of ethanol
was close to that of control experiments using nitrogen gas
(Fig.4 dotted line). In contrast, the amount of ethanol aer
the propane hydroxylation (70 mM) was not very different
from that of contamination from the reagents (70 mM),
suggesting that the side reaction producing background
ethanol competes with the propane hydroxylation reaction.
8
9
Q.-S. Li, J. Ogawa and S. Shimizu, Biochem. Biophys. Res.
Commun., 2001, 280, 1258–1261.
C. R. Otey, G. Bandara, J. Lalonde, K. Takahashi and
F. H. Arnold, Biotechnol. Bioeng., 2006, 93, 494–499.
0 C. J. C. Whitehouse, W. Yang , J. A. Yorke, B. C. Rowlatt,
A. J. F. Strong, C. F. Blanford, S. G. Bell, M. Bartlam,
1
L.-L. Wong and Z. Rao , ChemBioChem, 2010, 11, 2549–2556. 30 The background 2.9 mM methanol was also observed in the
1
1
1
1 M. Dietrich, T. A. Do, R. D. Schmid, J. Pleiss and
V. B. Urlacher, J. Biotechnol., 2009, 139, 115–117.
ethane hydroxylation under the same reaction conditions as
for the methane hydroxylation.
2 P. Meinhold, M. W. Peters, M. M. Y. Chen, K. Takahashi and 31 Our reaction conditions are not completely identical to those
F. H. Arnold, ChemBioChem, 2005, 6, 1765–1768.
3 S. Kille, F. E. Zilly, J. P. Acevedo and M. T. Reetz, Nat. Chem.,
reported by Zilly et al., for instance, they used His-tag
P450BM3 and thus purication methods of P450BM3 are
different. In addition, purities of protein and chemicals
including gaseous alkanes would be different.
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