Highly efficient hydroxylation of gaseous alkanes at reduced temperature catalyzed by cytochrome P450BM3 assisted by decoy molecules

Article abstract of DOI:10.1142/S1088424615500145

Cytochrome P450BM3 functions as a small-alkane hydroxylase upon the addition of perfluorocarboxylic acids (PFs) as decoy molecules. The coupling efficiency (product formation rate per NADPH consumption rate) for the hydroxylation of small alkanes was improved by reducing the reaction temperature to 0°C.

Full text of DOI:10.1142/S1088424615500145

Journal of Porphyrins and Phthalocyanines
J. Porphyrins Phthalocyanines 2015; 19: 329–334
DOI: 10.1142/S1088424615500145
Published at http://www.worldscinet.com/jpp/
Highly efficient hydroxylation of gaseous alkanes
at reduced temperature catalyzed by cytochrome P450BM3
assisted by decoy molecules
Norifumi Kawakamia, Zhiqi Congb, Osami Shoji*b◊ andYoshihito Watanabe*c
a Department of Biosciences and Informatics, Keio University, 3-14-1 Hiyoshi, 223-8522 Yokohama, Japan
b Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku,
Nagoya, 464-8602, Japan
c Research Center for Materials Science Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan
Dedicated to Professor Shunichi Fukuzumi on the occasion of his retirement
Received 9 November 2014
Accepted 4 December 2014
ABSTRACT: Cytochrome P450BM3 functions as a small-alkane hydroxylase upon the addition of
perfluorocarboxylic acids (PFs) as decoy molecules. The coupling efficiency (product formation rate
per NADPH consumption rate) for the hydroxylation of small alkanes was improved by reducing the
reaction temperature to 0°C.
KEYWORDS: cytochrome P450, gaseous alkanes, perfluorocarboxylic acids, decoy molecules.
group and Zilly et al. recently reported that even wild-
INTRODUCTION
type P450BM3 can catalyze the hydroxylation of small
Hydroxylation of small alkanes catalyzed by enzymes
alkanes in the presence of a series of perfluorocarboxylic
has been recognized as an important process for
acids (PFs) with alkyl chain lengths from 5 to 14 (PFC5–
producing precursors of fine chemicals and feedstocks.
14); the latter perfluorinated compounds were named
Several small alkane hydroxylases, such as methane
“decoy molecules” (Fig. 1b, right) [12, 13]. According
monooxygenase and omega alkane hydroxylase AlkB,
to the reaction mechanism of fatty acid hydroxylation
have been widely studied in the context of industrial
by P450BM3 (Fig. 1b, left), fatty acid binding induces
applications [1–6]. In addition, a variety of engineered
a positive redox potential shift of the heme iron by
enzymes that catalyze the hydroxylation of small alkanes
removing a ligated water molecule, which is crucial for
has also been successfully prepared by site-directed and/
initiating the catalytic cycle. Molecular oxygen is then
or random mutagenesis [7, 8]. For example, cytochrome
reductively activated by utilizing two electrons from
P450BM3, which catalyzes the hydroxylation of
fatty acids having an alkyl chain length from 12 to 20
NADPH to form an oxoferryl(IV) porphyrin p cation
radical (so-called Compound I), which is responsible for
(C12–20) carbon units, has been engineered to catalyze
the hydroxylation of substrates (Fig. 1) [14–17].We found
the hydroxylation of gaseous alkanes such as ethane,
that because PFs have structures that are similar to those
propane, and butane [9–11]. Although wild-type
of fatty acids, it is also possible to initiate the catalytic
P450BM3 selectively catalyzes the hydroxylation of
cycle to form Compound I [12]; however, the PFs are
long-alkyl-chain fatty acids (Fig. 1b, left), our research
not hydroxylated because of the high dissociation energy
of C–F bonds. By adding small alkanes to the reaction
mixture, the produced Compound I can hydroxylate the
◊ SPP full member in good standing
small alkanes instead of PFs (Fig. 1b, right). Interestingly,
*Correspondence to: Osami Shoji, email: shoji.osami@a.mbox.
the catalytic activities of the alkane hydroxylation
depend on the alkyl chain length of the PFs. For example,
nagoya-u.ac.jp; Yoshihito Watanabe, email: p47297a@nucc.
cc.nagoya-u.ac.jp, tel: +81 52-789-3557, fax: +81 52-789-2953
Copyright © 2015 World Scientific Publishing Company

330
N. KAWAKAMI ET AL.
Fig. 1. (a) The active site of P450BM3 containing palmitoleic acid (left; PDB code: 1FAG) and the plausible active-site structure of
P450BM3 containing PFC10 and propane (right). (b) General reaction mechanism of P450BM3 (left) and the reaction mechanism
of propane hydroxylation catalyzed by P450BM3 with perfluorodecanoic acid (PFC10) as a decoy molecule (right). In the presence
of PFC10, although uncoupling reaction produces water molecule (dotted line in the reaction cycle), propane is converted to
2-propanol (solid line in the reaction cycle)
the highest catalytic activities for propane and butane
hydroxylation reactions were observed in the presence of
PFC10 and PFC9, respectively. Therefore, we concluded
that the PFs partially occupy the active site of P450BM3,
leaving a small amount of space that was suitable for
accommodating small alkanes (Fig. 1a, right). Although
small alkanes are hydroxylated in the presence of PFs, the
coupling efficiency (product formation rate per NADPH
consumption rate) was low: 18 and 55% for propane and
butane hydroxylation reactions, respectively. As reported
previously [18], Compound I, generated from P450BM3
in the presence of PFs, is also reduced back to the resting
state through a further two-electron reduction leading to
the production of H2O. This unproductive consumption
of Compound I competes with the hydroxylation of
small alkanes (Fig. 1b, right) [12, 19]. By increasing the
propane pressure from 8 kPa to 0.5 MPa, the coupling
efficiency was improved to 50% [19], indicating that the
concentration of small alkanes is a key parameter that
affects coupling efficiency. We reasoned that the coupling
efficiency could be improved if the enzymatic reaction
was performed at lower temperature because this would
lead to an increase in the concentration of gaseous alkanes
in the aqueous solution. We herein report that the effect
of temperature on the catalytic activity and coupling
efficiency of propane and butane hydroxylation reactions
catalyzed by wild-type P450BM3 in the presence of PFs
is indeed significant.
EXPERIMENTAL
Materials
P450BM3 was prepared according to the method
previously reported [12]. The concentration of the
enzyme was determined by CO-difference spectra. The
details of purification of P450BM3 are shown in below.
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HIGHLY EFFICIENT HYDROXYLATION OF GASEOUS ALKANES AT REDUCED TEMPERATURE CATALYZED
331
E. coli cells expressing P450BM3 suspended in 20 mM
1 min. One hour later, the amount of remaining NADPH
was estimated by measuring the absorbance at 340 nm.
Tris-HCl (pH 7.4) are disrupted by using an ultrasonicator
at 4°C. After removing cell debris by centrifugation, the
supernatant was applied to DE-52 column (Whatman).
The proteins weakly bound to DE-52 are washed out
with 20 mM Tris-HCl containing 50 mM KCl (pH 7.4)
and tightly bound proteins including P450BM3 are
eluted with the Tris-buffer containing 250 mM KCl. The
P450BM3 fraction was collected and further purified by
DEAE 650S (TOSOH). P450BM3 was eluted with the
Tris-HCl buffer with KCl concentration gradient from
0 to 120 mM. To remove the bound substrates, NADPH
was added immediately before applying to gel filtration
column, Sephacryl S-300 (GE Healthcare), equilibrated
with 20 mM Tris buffer with 100 mM KCl (pH 7.4)
and the P450BM3 fraction was collected. All chemical
reagents were purchased from commercial sources and
used without further purification. o-Cresol, m-cresol, and
perfluorononanoicacid(PEC9)wereobtainedfromTokyo
Chemical Industry Co. (Tokyo, Japan). Benzylalcohol,
p-cresol, and perfluorooctanoic acid (PFC8) were
purchased from Sigma-Aldrich Co., (St. Louis, MO).
Toluene were obtained from Kanto Chemical Co., Inc.
(Tokyo, Japan). The following chemicals were purchased
from WAKO Pure Chemical Industries, Ltd (Osaka,
Japan): benzene, and perfluorodecanoic acid (PFC10).
Gaseous alkanes are purchased from Taiyo Nippon Sanso
Corp. NADPH was purchased from Nakalai tesque, Inc.
Propanol and butanol detection
After one-hour reaction, 1 mL of reaction mixture was
transferred to new glass vial containing 0.15 g of sodium
nitrite for derivatization, and then 1 mM 3-pentanol was
added as internal standard. Before putting the sample on
ice, 1 mL of hexane (for propanol) or dichloromethane
(for butanol) were added. To the resulting reaction
mixture, 150 mL of aqueous solution of sulfuric acid
(20%) was gently added. The glass vial was kept on ice
for 15 min. The derivatized products, 2-propylnitrite
or 2-butylnitrite, were extracted and analyzed by Gas
Chromatography (GC). The analytical conditions were as
follows: column temperature, 323 K (holding 5 min) to
523 K (25 K/min, holding 3 min); the injection port and
FID detector temperature were 523 K; carrier gas, helium.
The products were identified based on the retention time
of authentic samples. The concentration of the products
was determined by using the ratio of peak area of the
product to the area of the internal standard (3-pentanol).
Hydroxylation of benzene
Benzene hydroxylation by P450BM3 was carried out
in 20 mM Tris-HCl (pH = 7.4) buffer containing 100 mM
KCl at 0°C for 1 h in the presence of 0.5 mM P450BM3,
10 mM benzene, 5 mM NADPH, and 100 mM PFC9.
PFC9 was dissolved in DMSO and added to the reaction
mixture. After 1 h reaction, a solution of hydrochloric
acid (1 M) was added to the reaction mixture to quench
the reaction followed by neutralization with a solution
of KOH (1 M). The resulting solution was filtrated and
analyzed by reverse phase HPLC. The HPLC analytical
conditions were as follows: flow rate, 0.5 mL/min;
acetonitrile/water = 1/1; column temperature, 30°C;
monitoring absorption at 210 and 270 nm. Phenol
was identified using authentic samples. Reaction was
performed at least three times.
Measurement
UV-vis spectra were recorded on a Shimadzu UV-2400
PC spectrophotometer. GC analysis was performed by
GC2014 (Shimadzu Corp.) with Rtx-1 column (Restek
Corp.). HPLC analysis was performed using an Inertsil®
ODS-3 column (4.6 mm × 250 mm; GL Sciences, Inc.,
Tokyo, Japan) installed on a Shimadzu SCL-10AVP
system controller equipped with Shimadzu LC-10ADVP
pump systems, a Shimadzu RF-10AXL fluorescence
spectrometer, a Shimadzu CTO-10AVP column oven,
and a Shimadzu DGU-12A degasser.
Hydroxylation of toluene
Gaseous alkane hydroxylation reactions
The reaction was carried out in the same manner as for
the hydroxylation of benzene. The reaction was evaluated
by reverse phase HPLC. Products were identified using
authentic samples.
Reaction mixture containing 500 nM P450BM3,
100 mM PFs was prepared in the gaseous alkane saturated
buffer at 20 or 0°C. The reaction temperature was kept
by water bath and iced water for the reaction at 20 and
0°C, respectively. PFs for stock solution were dissolved
in DMSO and adjusted to 20 mM. Gaseous alkane
saturated buffer was also prepared at each temperature.
Immediately after adding 5 mM NADPH (for the long-
term reaction, 20 mM of NADPH was used), the reaction
mixture was pressured by a balloon containing gaseous
alkane and molecular oxygen with the ratio of 4 to 1.
The remaining air in the reaction vessel was replaced by
making gas flow from balloon to the out via the vessel for
NADPH consumption
Immediately after preparing reaction mixture containing
0.5 mM P450BM3, 10 mM benzene, and 100 μM
perfluorinated carboxylic acids, reaction was initiated
by the addition of 5 mM NADPH. The absorbance of
NADPH at 340 nm was monitored. The concentration
of the NADPH was estimated using its molar extinction
-1
coefficient at 340 nm, 6220 M-1 cm .
.
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N. KAWAKAMI ET AL.
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HIGHLY EFFICIENT HYDROXYLATION OF GASEOUS ALKANES AT REDUCED TEMPERATURE CATALYZED
333
Table 2. The catalytic activity of propane hydroxylation by
RESULTS AND DISCUSSION
P450BM3 with PFC10 at 0 and 20°C for 24 h. The amount of
2-propanol and NADPH consumed after 24 h reaction are given
in [mM]. The total tuenover number (TON) and the coupling
efficiency are given in nmol/nmol-P450BM3, and [Product]/
[NADPH consumed] × 100, respectively
We first investigated whether the use of P450BM3
in conjunction with PFs catalyzed the hydroxylation of
propane and butane gas at 0°C (Table 1) and confirmed
that, even at this low temperature, the P450BM3–
PF complexes functioned as a monooxygenase to
selectively yield 2-propanol and 2-butanol, respectively.
To understand the effect of reaction temperature,
hydroxylation of propane and butane was also carried out
at 20°C and the coupling efficiency and hydroxylation
rate were compared with those measured at 0°C [20].
The coupling efficiency of propane hydroxylation
improved slightly from 38 to 44% in the presence of
PFC10, upon reducing the reaction temperature, whereas
the hydroxylation rate decreased from 3240 to 960
nmol/h/nmol-P450BM3. Similarly, a small improvement
in the coupling efficiency and a decrease in the catalytic
activity were observed for butane hydroxylation in the
presence of PFC10 at 0°C. The coupling efficiency for
propane hydroxylation upon lowering the temperature
showed a more significant improvement, from 31 to
57%, in the presence of PFC9. Furthermore, the coupling
efficiency of butane hydroxylation was also drastically
improved at lower temperature, from 78 to 96%, in the
presence of PFC9. Notably, butane hydroxylation with
PFC9 at 0°C resulted in almost complete coupling
(96%) while maintaining a catalytic activity comparable
to that measured for the reaction performed at 20°C.
The uncoupling reaction in the absence of alkane
gas was also dramatically suppressed, from 4200 to
820 turnover frequency, by lowering the temperature
(Table 1), suggesting that P450BM3 was activated when
the PFC9 and gaseous alkanes occupied the active-site
space simultaneously. Although the uncoupling reaction
in the absence of alkane gas was also suppressed in the
case of PFC10 (from 7720 to 1320 turnover frequency),
the coupling efficiency was not much improved. We
reasoned that PFC10 is too effective for initiating the
generation of active species even at 0°C. Because PFC10
alone was sufficient enough for the generation of active
species, the uncoupling reaction was predominant even
in the presence of alkane gas.
2-propanol,
μM
NADPH
consumption, efficiency,
Coupling Turnover
number
(TON)
μM
%
20°C
0°C
2380
4920
14100
12400
17
40
4760
9840
Given that the solubility of gaseous molecules is
inversely proportional to solvent temperature, the
concentration of gaseous molecules in the buffer solution
increases upon lowering the reaction temperature and
thus the coupling efficiency is improved by performing
the reaction at 0°C rather than at 20°C. In fact, the
solubilities of propane and butane in water at 4°C are
reported to be 3.46 and 3.21 M, respectively, which are
twice as large as those of propane at 19.8°C (1.74 M) and
butane at 20°C (1.46 M) [21]. In addition to these results,
propane hydroxylation in the presence of PFC9 over a
longer period showed a total turnover number (TON) of
9840, with 40% coupling efficiency for 24 h reaction at
0°C, whereas the same reaction performed at 20°C had
a TON of 4760, with 18% coupling efficiency, indicating
that cooling the reaction mixture was also effective with
respect to the total amount of product formed (Table 2).
The enantioselectivity for the formation of 2-butanol
increased slightly, especially in the presence of PFC9
(from 14 to 20% ee, Table 1), but the small difference
suggests that the orientation of butane in the active site
is not significantly affected by the reaction temperature.
Although improved butane hydroxylation by engineered
P450BM3 at 0°C was recently described, the effect of
temperature upon the enantioselectivity was not reported
[22]. To investigate the effect of performing the reaction at
0°C upon hydroxylation of substrates other than gaseous
alkane, we also conducted the hydroxylation reaction with
benzene and toluene [23] at 0 and at 20°C for 1 h (Table 3).
Table 3. The catalytic activity of benzene and toluene hydroxylations by P450BM3 with PFC9 at 0°C and 20°C for 1 h. The
amount of products after 1 h reaction are given in [mM]. The amount of NADPH consumed and the coupling efficiency are given
in [mM] and [Product]/[NADPH consumed] × 100, respectively
Substrate
Product, mMa
NADPH consumption, mMa Coupling efficiency, %
Major
Minor
Benzene 0°C
Benzene 20°C
Toluene 0°C
Toluene 20°C
980 180 (Phenol)
2030 480 (Phenol)
1830 70 (o-Cresol)
3070 20
8270 420
4200 140
9860 30
32
25
50
47
6
7
4
3
280 30 (Benzyl alcohol)
4100 260 (o-Cresol) 510 50 (Benzyl alcohol)
aValues are averages standard deviation calculated from at least three different experiments.
Copyright © 2015 World Scientific Publishing Company
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N. KAWAKAMI ET AL.
The coupling efficiency for the hydroxylation of benzene
(25%) and toluene (47%) at 20°C was improved to 32%
and50%,respectively,byreducingthetemperatureto0°C.
Because the regioselectivity of toluene hydroxylation was
not affected significantly by the reaction temperature,
we presume the orientation of toluene in the cavity of
P450BM3 at 0°C would be not much different from that
at 20°C. The improved coupling efficiency observed for
the benzene and toluene hydroxylation using PFC9 as a
decoy molecule could be attributed to suppression of the
uncoupling reaction in the absence of substrate at 0°C.
Giventhatthecouplingefficiencywasclearlyimproved
with gaseous alkane hydroxylation, the improved
solubility of gaseous alkanes at 0°C in the buffer solution
together with the suppression of the uncoupling reaction
in the absence of substrate at 0°C contribute to efficient
reactions catalyzed by P450BM3 with the assistance of
decoy molecules.
3. Koch DJ, Chen MM, van Beilen JB and Arnold FH.
Appl. Environ. Microbiol. 2009; 75: 337–344.
4. Nieder M and Shapiro J. J. Bacteriol. 1975; 122:
93–98.
5. Staijen IE, van Beilen JB and Witholt B. Eur. J.
Biochem. 2000; 267: 1957–1965.
6. JiangY and Dalton H. Biochim. Biophys. Acta, Gen.
Subj. 1994; 1201: 76–84.
7. Bell SG, Stevenson JA, Boyd HD, Campbell
S, Riddle AD, Orton EL and Wong LL. Chem.
Commun. 2002; 490–491.
8. Xu F, Bell SG, Lednik J, InsleyA, Rao ZH andWong
LL. Angew. Chem. Int. Ed. 2005; 44: 4029–4032.
9. Boddupalli SS, Pramanik BC, Slaughter CA,
Estabrook RW and Peterson JA. Arch. Biochem.
Biophys. 1992; 292: 20–28.
10. Meinhold P, Peters MW, Chen MMY, Takahashi K
andArnold FH. ChemBioChem 2005; 6: 1765–1768.
11. Fasan R, Meharenna YT, Snow CD, Poulos TL and
Arnold FH. J. Mol. Biol. 2008; 383: 1069–1080.
12. Kawakami N, Shoji O and Watanabe Y. Angew.
Chem. Int. Ed. 2011; 50: 5315–5318.
13. Zilly FE, Acevedo JP, Augustyniak W, Deege A,
Hausig UW and Reetz MT. Angew. Chem. Int. Ed.
2011; 50: 2720–2724.
14. Rittle J and Green MT. Science 2010; 330: 933–937.
15. Makris TM, von Koenig K, Schlichting I and Sligar
SG. J. Inorg. Biochem. 2006; 100: 507–518.
16. Murataliev MB and Feyereisen R. Biochemistry
1996; 35: 15029–15037.
CONCLUSION
We have demonstrated that the coupling efficiency
of propane and butane hydroxylation catalyzed by
the P450BM3-decoy molecule system is significantly
improved by performing the reaction at 0°C.The coupling
efficiency for propane and butane reached 57 and 96%,
respectively. Conducting the reaction at low temperature
is beneficial not only for the coupling efficiency but also
for the total amount of product formed through small
alkane hydroxylation. Although the turnover rate at
0°C clearly decreased, performing the reaction at 0°C
could be one of the strategies to improve the efficiency
of gaseous alkane hydroxylation catalyzed by P450BM3
with the assistance of decoy molecules.
17. Perera R, Sono M, Raner GM and Dawson JH.
Biochem. Biophys. Res. Commun. 2005; 338:365–371.
18. Pons N, Pipino S and De Matteis F. Biochem.
Pharmacol. 2003; 66: 405–414.
19. Kawakami N, Shoji O and Watanabe Y. Chem. Sci.
2013; 4: 2344–2348.
20. We found that air was still remained in the reaction
vessel in our previous method [12]. Incomplete
replacement of air with small alkane causes the
low concentration of small alkanes in the reac-
tion mixture. By thoroughly replacing the remain-
ing air by small alkanes, we obtained the higher
coupling efficiency of propane and butane hydrox-
ylation than those of the previous results [12].
21. Yalkowsky SH, He Y and Jain P. Handbook of
Aqueous Solubility Data, 2nd ed., CRC Press: Boca
Raton, 2010; pp 75 and 127.
Acknowledgements
This work was supported by Grants-in-Aid for
Scientific Research (S) toY.W. (24225004), Grant-in-Aid
for Scientific Research on Innovative Areas “Molecular
Activation Directed toward Straightforward Synthesis” to
O.S. (25105724), and a Grant-in-Aid forYoung Scientists
(A) to O.S. (21685018) from the Ministry of Education,
Culture, Sports, Science, and Technology (Japan).
REFERENCES
22. Staudt S, Muller CA, Marienhagen J, Boing
C, Buchholz S, Schwaneberg U and Groger H.
Beilstein Journal of Organic Chemistry 2012; 8:
186–191.
1. Merkx M, Kopp DA, Sazinsky MH, Blazyk JL,
Muller J and Lippard SJ. Angew. Chem. Int. Ed.
2001; 40: 2782–2807.
23. Shoji O, Kunimatsu T, Kawakami N and Watanabe
Y. Angew. Chem. Int. Ed. 2013; 52: 6606–6610.
2. Lee SJ, McCormick MS, Lippard SJ and Cho US.
Nature 2013; 494: 380–384.
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