Monoterpene hydroxylation with an artificial self-sufficient P450 utilizing a P450SMO reductase domain for the electron transfer

Article abstract of DOI:10.1016/j.molcatb.2015.02.006

Cytochrome P450_SMO from Rhodococcus sp. ECU0066 is a natural self-sufficient P450 monooxygenase, consisting of a heme domain, a flavin-reductase domain containing FMN and NADPH binding sites, and a [Fe₂S₂] ferredoxin domain. P450_cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor. The variant P450_cam (Y96F/V247L) was reported for the oxidation of monoterpene by protein engineering. In this work, we constructed an artificial self-sufficient P450-type monoterpene hydroxylase by connecting the P450_SMO reductase domain and the P450_cam (Y96F/V247L) domain together with a linker region (G₄S)₄. The resultant chimeric P450 enzyme could catalyze the hydroxylation of (-)-limonene and α-pinene as well as camphor, which were all inactive for the natural fusion protein P450_SMO. Co-expression of the fused P450 with a glucose dehydrogenase (GDH) improved the (-)-limonene conversion as sufficient NADPH was regenerated in the system with glucose as a cosubstrate. This work illustrated that P450_SMO reductase might act as an electron donor partner of P450s and might be used for fusion with heterogeneous P450 domains to elucidate the catalytic function of other unknown P450s.

Full text of DOI:10.1016/j.molcatb.2015.02.006

Journal of Molecular Catalysis B: Enzymatic 116 (2015) 78–82
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Monoterpene hydroxylation with an artificial self-sufficient P450
utilizing a P450SMO reductase domain for the electron transfer
∗
∗∗
Zheng-Jiao Luan, Yue-Cai Yin, Ai-Tao Li, Hui-Lei Yu , Jian-He Xu
State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Centre for Biomanufacturing Technology, East China University of
Science and Technology, 130 Meilong Road, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytochrome P450SMO from Rhodococcus sp. ECU0066 is a natural self-sufficient P450 monooxygenase,
Received 23 September 2014
Received in revised form 10 February 2015
Accepted 10 February 2015
Available online 20 February 2015
consisting of a heme domain, a flavin-reductase domain containing FMN and NADPH binding sites, and a
[Fe2S2] ferredoxin domain. P450cam catalyzes the hydroxylation of camphor to 5-exo-hydroxycamphor.
The variant P450cam (Y96F/V247L) was reported for the oxidation of monoterpene by protein engineering.
In this work, we constructed an artificial self-sufficient P450-type monoterpene hydroxylase by connect-
ing the P450SMO reductase domain and the P450cam (Y96F/V247L) domain together with a linker region
Keywords:
(
G4S)4. The resultant chimeric P450 enzyme could catalyze the hydroxylation of (−)-limonene and ˛-
Self-sufficient P450s
Monoterpene hydroxylation
Electron transfer
pinene as well as camphor, which were all inactive for the natural fusion protein P450SMO. Co-expression
of the fused P450 with a glucose dehydrogenase (GDH) improved the (−)-limonene conversion as suf-
ficient NADPH was regenerated in the system with glucose as a cosubstrate. This work illustrated that
P450SMO reductase might act as an electron donor partner of P450s and might be used for fusion with
heterogeneous P450 domains to elucidate the catalytic function of other unknown P450s.
Protein engineering
©
2015 Elsevier B.V. All rights reserved.
1
. Introduction
Even the reducing equivalents are successfully transferred to the
heme domain, they can be employed in the formation of water or
hydrogen peroxide and are therefore useless for substrate oxidation
[5,6].
P450s are hemoproteins encoded by a super-family of genes.
They can convert a broad variety of substrates and catalyze a series
of interesting chemical reactions. It is worth mentioning that P450s
can participate in hydroxylation of terpenes, which are essential
intermediates for the spices industry [1]. The cytochrome P450
reactions must be associated with electron donor partner pro-
tein(s) for two electrons transferred from nicotinamide adenine
dinucleotide phosphate [NAD(P)H] to the heme domain of a P450.
For example, adrenal mitochondrial P450 systems obtain electrons
from NADPH via adrenodoxin reductase and adrenodoxin; and the
liver microsomal P450s obtain electrons from NADPH via a FAD
and FMN-containing P450 reductase [2]. The inherent requirement
for the individual protein partner(s) usually made P450s difficult
for significant discovery and application [3,4]. Meanwhile, elec-
trons generated by the redox partner, which are intended to fuel
the P450 oxidation process, are not always transferred effectively.
P450BM3, which is naturally fused to a eukaryotic-like reduc-
tase, represents an effective solution to these limitations. The fusion
nature of this enzyme greatly improves the electron-transfer effi-
ciency and the oxidation activity for fatty acids [7]. Based upon the
self-sufficiency of this naturally fused enzyme, a number of engi-
neered proteins of diverse eukaryotic P450s bearing a reductase
domain from P450BM3 have been generated with in vitro activities
[8–10]. This provides ready access to the great catalytic versatil-
ity of the membrane-bound eukaryotic P450s. Recently, it has been
reported that the reductase domain of P450RhF from Rhodococcus
sp. NCIMB 9784 can be fused to some Class I P450s or Class II
eukaryotic microsomal P450s (P450cam, P450bzo, P450balk, P450PikC
,
P450XplA, CYP73A5) for the generation of self-sufficient enzymes
in whole cell biotransformation or in cell free assays [11–15].
P450SMO, a new member of CYP116 family, has been discovered in
our laboratory, showing significant sulfoxidation activity toward
several sulfides [16,17]. P450SMO, belonging to Class IV P450 fam-
ily, contains a heme domain, FMN and NADPH binding motifs and a
∗
Corresponding author. Tel.: +86 2164250840; fax: +86 21 64250840.
Corresponding author. Fax: +86 21 64250840.
E-mail addresses: huileiyu@ecust.edu.cn (H.-L. Yu), jianhexu@ecust.edu.cn
∗
∗
[
Fe S ] ferredoxin-like center. The composition of P450
reduc-
2
2
SMO
tase domain has the similarity to the electron transfer partner of
(
J.-H. Xu).
http://dx.doi.org/10.1016/j.molcatb.2015.02.006
381-1177/© 2015 Elsevier B.V. All rights reserved.
1

Z.-J. Luan et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 78–82
79
Scheme 1. Construction of an artificial P450 system: P450cam (Y96F/V247L)–P450SMO red.
Class I P450. So we speculated whether the P450SMO reductase
domain could act as an effective redox partner for generating effi-
cient P450 chimeras.
(pH 7.5), and disrupted by sonification. After centrifugation, the
soluble fractions were subjected to spectrophotometric analysis or
reductase domain activity assay.
Cytochrome P450cam (PdR, Pdx) catalyzes the oxidation of the
bicyclic compound d-(+)-camphor to 5-exo-hydroxycamphor, the
first step in the camphor metabolism pathway of the soil bacterium
Pseudomonas putida. The Y96F/V247L mutant of P450cam could cat-
alyze the hydroxylation of (−)-limonene in one step, producing
2
.3. Spectrophotometric analysis and reductase activity assay of
P450a F1–F4
To quantitate the content of active cytochrome P450 in the
(
−)-trans-isopiperitenol, (−)-trans-carveol and (−)-cis-limonene
recombinant E. coli, the soluble fraction of P450a F1–F4 were used
to measure the CO-reduced P450 absorption with a UV-vis spec-
trophotometer [19]. The reductase activity was determined by
measuring the increase in absorbance at 550 nm due to the reduc-
tion of cytochrome c [20].
epoxide [18]. In the present work, we therefore constructed a
self-sufficient chimera P450cam (Y96F/V247L)–P450SMO red, by fus-
ing the P450SMO reductase domain with the P450cam mutant
domain together (see Scheme 1). By optimizing the linker sequence
between the two domains and co-expressing with glucose dehy-
drogenase (GDH, used for NADPH regeneration), the whole cell
could be used for monoterpene hydroxylation, indicating that the
P450SMO reductase might be used as the electron transfer partner
for heterologous P450s. Furthermore, the biosynthetic P450s (Class
I) lacking such a universal reductase may be engineered similarly
into diverse self-sufficient P450s for either functional characteri-
zation or potential application.
2
.4. Bioconversion of (−)-limonene by the fused enzymes with
different linkers
To determine the catalytic activity of the fused enzymes with
different linkers, we performed a 1 mL reaction with the cell free
extracts. The reaction mixtures containing 50 mM potassium phos-
phate buffer (pH 7.5), 1 ␮M P450a F1–F4, 100 ␮M (−)-limonene and
◦
1
00 ␮M NADPH were incubated at 25 C. After incubation for 12 h,
2
. Experimental
the reaction mixture was extracted with 900 ␮L ethyl acetate by
vigorous shaking. The organic layer was separated by centrifuga-
tion, dried over anhydrous sodium sulfate, and then subjected to
GC–MS analysis.
2.1. Engineering the chimeric P450cam (Y96F/V247L)–P450SMO red
F1–F4
The negative control was conducted under the same condition
using the same amount P450cam (Y96F/V247L) and the P450SMO
reductase, which were not fused together. The positive control was
conducted by P450cam system under the same condition, which
contained P450cam (Y96F/V247L), putidaredoxin reductase (PdR)
and putidaredoxin (Pdx).
The genes of P450cam and P450SMO were cloned from P. putida
PpGl (ATCC17453) and Rhodococcus sp. ECU0066, respectively. Site-
directed mutagenesis of P450cam (Y96F/V247L) gene was carried
out with the Quick Change® Site-directed Mutagenesis Kit. The
primers for the mutagenesis were shown in Table S1 (Supporting
Information).
As shown in Table S1, P450cam (Y96F/V247L) and P450SMO
reductase were amplified with primers A, C1/D1, C2/D2, C3/D3,
C4/D4, B to construct four different linkers. The amplified P450 gene
2.5. Plasmid construction for co-expression of P450cam
(Y96F/V247L)–P450SMO red F3 and GDH genes
ꢀ
ꢀ
has NdeI site at 5 -ends and HindIII at the 3 -ends. Meanwhile, the
ꢀ
ꢀ
P450SMO red gene has HindIII site at 5 -ends and XhoI at the 3 -ends.
Then the two genes were ligated into pET28a(+) and fused together.
The fused enzymes P450cam (Y96F/V247L)–P450SMO red F1–F4 have
the His-tag at the N-terminal for subsequent purification. The fused
enzymes are abbreviated as P450a F1–F4 in this article.
Plasmids and the primers used for co-expression in this study
are listed in Tables S2 and S3. For construction of the co-expression
system, the glucose dehydrogenase (GDH) gene was amplified with
primers 1 and 2 using genome DNA from Bacillus megaterium. The
resulting 786 bp fragment was digested with NdeI and XhoI and
then ligated into pACYCDuet-1 which was digested with the same
restriction enzymes, generating the plasmid pACYCDuet-1-GDH.
Successful ligation into pACYCDuet-1 was confirmed by restriction
analysis.
2.2. The expression of fusion enzymes
Recombinants containing pET28a(+)-P450a F1–F4 were grown
in 100 mL Luria-Bertani (LB) medium with 100 ␮g/mL of kanamycin
at 37 C. After the optical density OD600 had reached 0.5, 0.2 mM
Co-expression of P450a F3 and GDH genes was performed
in a two-plasmid system with different origins for replication,
P450a and GDH genes were respectively cloned into pET28a(+)
and pACYCDuet-1. The same E. coli strain transformed with both
pET28a(+)-P450a and pACYCDuet-1-GDH was named as BL21-
pET28a-P450a F3/pACYCDuet-GDH (see Scheme 2).
◦
isopropyl-ˇ-d-thiogalactopyranoside (IPTG) and 0.2 mM ı-amino
levulinic acid (ı-ALA) were added, and then the cultivation was
◦
continued with reciprocal shaking for 18 h at 25 C. The cells
were collected, suspended in 50 mM potassium phosphate buffer

8
0
Z.-J. Luan et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 78–82
Scheme 2. The dual plasmid configurations for co-expression of P450a F3 and GDH in E. coli.
2.6. Monoterpene hydroxylation with BL21-pET28a-P450a
3. Results and discussion
F3/pACYCDuet-GDH
3.1. Construction of fusion enzymes P450a F1–F4 with different
Biotransformation was performed in 10 mL reaction mixture
with a 50 mL sealed conical flask. The reaction mixtures contain-
ing 50 mM potassium phosphate buffer (pH 7.5), 0.1 g recombinant
wet cell/mL, 100 ␮M (−)-limonene (camphor or ˛-pinene), 100 ␮M
linkers
The successful construction of a recombinant fusion protein
requires two indispensable elements: the component proteins and
the linker [23]. Here based on the desired monoterpene hydroxyl-
ation of the fusion protein, we selected P450cam (Y96F/V247L) and
P450SMO red directly. On the other side, the selection of a suitable
linker to join the protein domains together is very crucial in this
work. We designed four different linkers for connecting P450cam
+
◦
NADP , 5 mM glucose were incubated at 25 C. After incuba-
tion for a certain time, samples were withdrawn for GC–MS
analysis.
2
.7. The dissociation constants (K ) of P450a F3 with
d
(Y96F/V247L) and P450SMO red domains with different length and
(
−)-limonene, camphor and ˛-pinene
secondary structure. Linkers F1 [(G S) ] and F3 [(G S) ] contain
4
3
4
4
flexible residues glycine and serine so that the adjacent protein
domains can move freely to close one another and maintain nec-
essary inter-domain interactions. Meanwhile we constructed the
The dissociation constants (K ) of P450a F3 were determined
d
◦
at 30 C by titration of 1 mL purified P450a F3 (1 ␮M) in 50 mM
phosphate buffer (pH 7.5) with substrates (−)-limonene, camphor
and ˛-pinene in ethanol. The spectroscopic changes (350–500 nm)
associated to the sequential addition of substrate (in the range of
relatively rigid linkers F2 [RLAST] and F4 [(RLAST) ] for a com-
2
parison. Fig. 1 shows the SDS-PAGE of the protein expression. The
production of active P450 protein and the activity of the reduc-
tase domain were on the same level for different fused enzymes
0
.1–35 ␮M) were recorded. The difference in absorbance between
the wavelength maximum (418 nm) and minimum (390 nm) was
plotted versus the substrate concentration into a Michaelis–Menten
equation to estimate Kd [21].
(Table 1).
As shown in Table 1, (−)-limonene biotransformations were
performed using P450a with different linkers (F1–F4). The reactions
2.8. Analytical methods
The analysis of products was performed by GC–MS (Shimadzu
GC–MS QP2010 Plus) with an intercap-5 column (30 m length;
0
2
.25 mm diameter). The injection and detection temperatures were
◦
◦
50 C and 230 C, respectively. The column was subjected to an ini-
◦
tial temperature of 50 C for 3 min, with the temperature then being
◦
◦
raised to 250 C at a rate of 10 C/min for 10 min. The MS spectrum
of product was the same as the literature [22]. (−)-Isopiperitenol
gave a 12.05 min peak and was quantified by a selective ion
monitoring mode generating standard curves. (−)-Carveol and
Fig. 1. SDS-PAGE analysis of the fused enzymes. Lanes 1, 3, 5 and 7 were the super-
natant of P450a F1–F4, respectively. Lanes 2, 4, 6 and 8 were the precipitant of the
fused enzymes.
(
−)-limonene epoxide showed the peaks at 12.30 and 10.95 min,
respectively.

Z.-J. Luan et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 78–82
81
Table 1
Activity assay of the fused enzymes.
F1 [(G4S)3]
F2 [RLAST]
F3 [(G4S)4]
F4 [(RLAST)2]
[
P450] (nmol/gwet cell)a
32.6 ± 0.3
9.76 ± 0.1
2.1 ± 0.1
40.4 ± 1.5
11.0 ± 0.2
54.6 ± 0.8
10.0 ± 0.5
5.2 ± 0.3
39.4 ± 1.1
11.4 ± 0.1
n.d.
b
Reductase activity (kU/gwet cell)
c
d
[(−)-Isopiperitenol] (␮M)
n.d.
a
Ferrous CO versus ferrous difference spectrum was used for the quantitation of the P450 (see Fig. S1).
The activity of reductase domain was measured by NADPH-cytochrome c reduction assay. One unit of enzyme activity was defined as the amount of enzyme catalyzing
b
the reduction of 1 nmol of cytochrome c per min.
c
The concentration of (−)-isopiperitenol was measured by GC–MS. The TIC and mass spectra for the products were shown in Fig. S2.
d
n.d.: no product was detected.
3.2. Co-expression of GDH with the fused enzyme P450a F3
As stated above, P450s require a cofactor to supply electrons for
the substrates oxygenation. However, the most common source
of reducing equivalents, NAD(P)H, has proven to be a major bar-
rier hampering the industrial implementation of P450s [24–26].
So we introduced the cofactor regeneration pathway into the E.
coli cell containing the P450a F3. Comparing the different co-
expression systems, the dual plasmid co-expression system was
chosen, which showed higher activity of GDH, as well as higher pro-
tein expression level of P450 (Table S4). As sufficient NADPH was
regenerated, the concentration of (−)-isopiperitenol was signifi-
cantly improved by approximately 10 folds upon the introduction
of GDH co-expressing system (Fig. 2). It suggested that NADPH was
limiting in the absence of the GDH regeneration system.
Fig. 2. Hydroxylation of (−)-limonene with or without GDH. Condition A: bio-
transformation with whole cells of BL21-pET28a(+)-P450a F3, plus 100 ␮M NADPH;
condition B: biotransformation with whole cells of the co-expression system BL21-
+
pET28a(+)-P450a F3/pACYCDuet-GDH, plus 100 ␮M NADP ; condition C: in vitro
biotransformation with the cell free extract of BL21-pET28a(+)-P450a F3, plus GDH
+
(
20 U) and 100 ␮M NADP .
3
.3. Biotransformation of (−)-limonene by BL21-pET28a-P450a
F3/pACYCDuet-GDH
were carried out using the same amount of P450s (1 ␮M). No
product was detected for the negative control, among the fused
enzymes, P450a F2 and F4 gave no hydroxyl product after 12 h.
The fused enzyme F1 and F3 both mediated the conversion of
limonene to isopiperitenol, and that the activity of F3 appeared
to be higher. Perhaps the appropriate length of the flexible linker
F3 [(G S) ] could provide the joined domains the chance for nec-
essary movement or interaction. While, the ␣-helix linkers may
act as a rigid spacer between protein domains and make the
independent structures that did not interact with the adjacent
protein domains. So the following experiments were all based on
P450a F3.
(−)-Limonene is the major component of peel oil from oranges
and lemons, so it is volatile like other terpenes. Thus high-yield
biotransformations should be performed with a closed system.
On the other hand, the hydroxylation reaction catalyzed by P450s
needs oxygen as the oxidant, thus the availability of oxygen in the
closed flask might have a significant influence on the hydroxyl-
ation. So we optimized the reaction system in flasks with different
loading volume, indicating that the loading of 10 mL cell suspen-
sion in a 50-mL closed conical flask was better (Fig. S3). Here, the
reaction was conducted using 0.1 g wet cell/mL buffer, 100 ␮M (−)-
4
4
+
limonene, 100 ␮M NADP , and 5 mM glucose. Under the optimum
condition, we got the reaction progress curve of the (−)-limonene
Table 2
The Kd values and product distribution of P450a F3 with different substrates.
Substrate
Kd0 (␮M)a
Kd (␮M)b
Product distribution (%)c
(−)-Limonene
8.6 ± 2.6
9.6 ± 1.4
46
38
16
Camphor
5.2 ± 1.0
6.5 ± 0.6
4.1 ± 0.8
3.6 ± 0.9
89
54
11
46
˛
-Pinene
a
b
c
The dissociation constants of P450cam (Y96F/V247L) toward (−)-limonene, camphor and ˛-pinene were shown in Fig. S4 (A).
The dissociation constants of purified P450a F3 toward (−)-limonene, camphor and ˛-pinene were shown in Fig. S4 (B).
Values were obtained from GC–MS peak area integrations. The oxidation products were analyzed by GC–MS. Fig. S5 gives the mass spectra of the products.
Not detected.
d

8
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Z.-J. Luan et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 78–82
to carry out oxidation reactions. The Class I or biosynthetic P450s
lack such a universal reductase partner and the discovery of their
natural partner may be very laborious. The chimeric technology we
presented here might be a solution to this problem. On the other
hand, because the P450s and their reductase domains are origi-
nated heterogeneously, the fused enzyme showed a relatively low
NADPH coupling efficiency (less than 10% toward (−)-limonene,
compared to 62% coupling efficiency of P450cam-PdR-Pdx system).
Co-expression of a glucose dehydrogenase in the recombinant E.
coli cell containing P450 gene could solve the above problem and
supply sufficient NADPH for the hydroxylation reaction. For fur-
ther study, we need to pay a close attention to the interactions
between the two heterogeneous domains and the improvement of
their coupling efficiency.
Fig. 3. Biotransformation of (−)-limonene by BL21-pET28a-P450a F3/pACYCDuet-
GDH.
Acknowledgements
This work was financially supported by the National Natu-
ral Science Foundation of China (No. 21276082) and the Ministry
of Science and Technology, P. R. China (Nos. 2011CB710800 and
biotransformation (Fig. 3) where the concentration of desired prod-
uct (−)-isopiperitenol was quantitatively measured by GC–MS.
2
012AA022201D).
3.4. Bioconversion of other substrates by BL21-pET28a-P450a
F3/pACYCDuet-GDH
Appendix A. Supplementary data
The catalytic activity of the artificial self-sufficient P450 toward
other terpenes was checked in the 10 mL reaction system. As shown
in Table 2, the fused enzyme P450a F3 exhibited the activity toward
the hydroxylation of these substrates. The Kd values of the purified
enzyme P450a F3 with different substrates were also measured,
which are similar to the mutant P450cam (Y96F/V247L). This indi-
cates that the attachment of the heterologous reductase domain
has no significant impact on the substrate binding with P450cam
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.molcatb.2015.02.006.
References
[
[
1] D. Werck-Rerchhart, R. Feyereisen, Genome Biol. 1 (2000) 3001–3009.
2] F. Hannemann, A. Bichet, K.M. Ewen, R. Bernhardt, Biochim. Biophys. Acta 1770
(
Y96F/V247L) domain.
(
2006) 330–344.
3] B. Schiffler, R. Bernhardt, Biochem. Biophy. Res. Commun. 312 (2003)
23–228.
[
2
4
. Conclusions
[
[
4] D.F.V. Lewis, P. Hlavica, Biochim. Biophys. Acta 1460 (2000) 353–374.
5] E. O’Reilly, V. Köhler, S.L. Flitsch, N.J. Turner, Chem. Commun. 47 (2011)
2490–2501.
In this report, we successfully constructed an artificial self-
[
6] J.B. Van Beilen, W.A. Duetz, A. Schmid, B. Witholt, Trends Biotechnol. 21 (2003)
sufficient P450 system (P450a), i.e., P450cam (Y96F/V247L)–
P450SMO red, utilizing the P450SMO reductase domain for the elec-
tron transfer. The fused enzyme could effectively hydroxylate
170–177.
[7] L.O. Narhi, A.J. Fulco, J. Biol. Chem. 262 (1987) 6683–6690.
[8] V.R. Dodhia, A. Fantuzzi, G. Gilardi, J. Biol. Inorg. Chem. 11 (2006) 903–916.
[
9] S. Eiben, H. Bartelmäs, V.B. Urlacher, Appl. Microbiol. Biotechnol. 75 (2007)
055–1061.
(
−)-limonene to (−)-isopiperitenol, (−)-carveol and (−)-limonene
1
epoxide, which are important intermediates for spices and pharma-
ceutical industry. Two major factors, the secondary structure and
the length of the linker between the P450cam (Y96F/V247L) domain
and the P450SMO reductase domain, have been varied to optimize
the fusion system. The results have shown that the linker modifica-
tion may alter electron transport or influence the extent to which
correctly folded fusion protein is formed. In our study, the flexible
loop linker, (G S) , could enable the two joined domains to move
[
[
[
10] M. Fairhead, S. Giannini, E.M.J. Gillam, G. Gilardi, J. Biol. Inorg. Chem. 10 (2005)
842–853.
11] M. Nodate, M. Kubota, N. Misawa, Appl. Microbiol. Biotechnol. 71 (2006)
455–462.
12] S.Y. Li, L.M. Podust, D.H. Sherman, J. Am. Chem. Soc. 129 (2007) 12940–12941.
[13] A. Robin, G.A. Roberts, J. Kisch, F. Sabbadin, G. Grogan, N. Bruce, N.J. Turner, S.L.
Flitsch, Chem. Commun. (2009) 2478–2480.
[
14] A. Robin, V. Köhler, A. Jones, A. Ali, P.P. Kelly, E. O’Reilly, N.J. Turner, S.L. Flitsch,
Beilstein J. Org. Chem. 7 (2011) 1494–1498.
4
4
[15] J. Schückel, E.L. Rylott, G. Grogan, N.C. Bruce, ChemBioChem 13 (2012)
2758–2763.
and interact, which is crucial for the electron transfer. Analysis of
the P450 concentration and the activity of the reductase domain
revealed that the fused enzyme P450cam (Y96F/V247L)–P450SMO red
[
16] A.T. Li, J.D. Zhang, J.H. Xu, W.Y. Lu, G.Q. Lin, Appl. Environ. Microbiol. 75 (2009)
51–556.
5
[17] J.D. Zhang, A.T. Li, Y. Yang, J.H. Xu, Appl. Microbiol. Biotechnol. 85 (2009)
615–624.
F3 retains the original characteristics of the two domains. Mean-
[18] S.G. Bell, R.J. Sowden, L.L. Wong, Chem. Commun. (2001) 635–636.
[19] T. Omura, R. Sato, J. Biol. Chem. 239 (1964) 2370–2378.
[20] F.P. Guengerich, M.V. Martin, C.D. Sohl, Q. Cheng, Nat. Protoc.
1245–1251.
while, the Kd values of the fused enzyme with different substrates
are similar to the wild type, indicating that the fused heterolo-
gous reductase domain has no significant influence on the substrate
binding with P450cam (Y96F/V247L) domain. Considering providing
sufficient cofactor NADPH, we constructed the co-expression sys-
4
(2009)
[
[
21] J.B. Schenkman, I. Jansson, in: I.R. Phillips, E.A. Shephard (Eds.), Methods in
Molecular Biology, Humana Press Inc., Totowa, NJ, 1998, pp. 25–34.
22] C. Bertea, M. Schalk, C.J.D. Mau, F. Karp, M.R. Wildung, R. Croteau, Phytochem-
istry 64 (2003) 1203–1211.
tem of GDH with P450a F3, which improved the hydroxylation by
+
[23] X.Y. Chen, J.L. Zaro, W.C. Shen, Adv. Drug Deliv. Rev. 65 (2012) 1367–1369.
1
0-fold due to the enzymatic regeneration of NADPH from NADP
[
[
24] S.Q. Pham, P.F. Gao, Z. Li, Biotechnol. Bioeng. 110 (2013) 363–373.
25] H. Schewe, B.A. Kaup, J. Schrader, Appl. Microbiol. Biotechnol. 78 (2008)
55–65.
at the cost of glucose.
P450-catalyzed reactions depend on the redox partner for gen-
eration of active ferryloxo species which is a key for these enzymes
[
26] R. Wichmann, D. Vasic-Racki, Biochem. Eng. Biotechnol. 92 (2005) 225–260.