Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

Article abstract of DOI:10.1016/j.bbrc.2012.01.040

Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k_cat of ～25min^-1 was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP²H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP²H but not D₂O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

Full text of DOI:10.1016/j.bbrc.2012.01.040

Biochemical and Biophysical Research Communications 418 (2012) 464–468
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc
Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct
hydride transfer
⇑
Rüdiger Kaspera, Tariku Sahele, Kyle Lakatos, Rheem A. Totah
Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 4 January 2012
Available online 18 January 2012
Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A kcat of
ꢀ25 minꢁ1 was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein
which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-
domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP2H
to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with
the reductase domain alone, a deuterium incorporation in the product from NADP2H but not D2O, and
no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site.
However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve
allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduc-
tion of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the
metabolism of endogenous substrates or xenobiotics.
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
found applications in biotechnology and in generating authentic
drug metabolites [5–7].
Cytochrome P450BM-3 (CYP102A1) of Bacillus megaterium is an
established model system to study mechanistic aspects of mamma-
lian cytochrome P450-mediated metabolism of xenobiotics and
endogenous compounds [1]. The natural fusion of the heme domain
with the cytochrome P450BM-3 reductase domain (BMR) enables
impressive catalytic activities reaching as high as 17,000 minꢁ1
(kcat) with arachidonic acid as a substrate [2]. The high catalytic
activity is attributed to the tight alignment of the three functional
domains, facilitating efficient hydride transfer from the cofactor
NADPH to FAD, a subsequent reduction of FMN by a proton coupled
electron transfer from FAD, and finally a one electron heme iron
reduction through a highly reactive FMN intermediate to initiate
the substrate oxidation cycle in the heme active site of P450BM-3
[3]. The mechanism of this cascade of redox reactions has been
intensively studied and reviewed elsewhere [3,4]. Cytochrome
P450BM-3 is known to hydroxylate various fatty acids, possibly
the natural substrates [5], but reactions such as oxidations (allylic
and benzylic), epoxidations (stereospecific), dealkylations, and
even the oxidation of methane have also been studied. This mani-
fold of reaction mechanisms have been of particular interest and
We set out to investigate P450BM-3 wild type (WT) and several
variants for their ability to oxidatively deformylate 1,2,3,4-tetrahy-
dro-napthaline-2-carbaldehyde similar to studies performed by
Roberts et al. [8]. Interestingly, no deformylated metabolites were
observed instead the only metabolite formed was the primary
alcohol a result of the direct reduction of the carbonyl moiety. Sev-
eral aldehydes substrates were investigated including methoxy
benzaldehyde (MBA) and the only metabolite formed was always
the alcohol.
Usually, reductions of aldehydes are associated with carbonyl
reductases such as aldo–keto reductase [9], and can be associated
with NADPH dependent reductions in human liver microsomes
[10]. Recently in reconstituted systems [11,12], a range of CYP-en-
zymes were able to reduce
a,b-unsaturated fatty acids such as 4-
hydroxy nonenal by a proposed radical mediated mechanism. In
this report, we establish that P450BM-3, and an activity enhancing
variant F87A/T268A, reduces aldehydes to alcohols and confirm
that the reduction occurs through a direct hydride transfer from
NADPH to the carbonyl.
2. Materials and methods
Abbreviations: P450, cytochrome P450; BM-3, Bacillus megaterium cytochrome
P450 102A1; CPR, cytochrome P450 reductase; BMR, P450BM-3 reductase domain;
BMP, heme domain of P450BM-3; MBA, p-methoxy-benzaldehyde; WT, wild type
enzyme.
2.1. Chemicals
⇑
Unless otherwise stated, reagents were purchased from Sigma–
Aldrich (St. Louis, MO) and culture media from Fisher Scientific
Corresponding author. Fax: +1 206 685 3252.
E-mail address: rtotah@u.washington.edu (R.A. Totah).
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2012.01.040

R. Kaspera et al. / Biochemical and Biophysical Research Communications 418 (2012) 464–468
465
(New Jersey, NJ). The P450BM-3 clone was a kind gift from Dr. Ar-
mand Fulco (University of CA at Los Angels). S-NADP2H and R-
NADP2H were synthesized by a modified method of Ottolina et al.
[13] and purified using an ion exchanger resin DE-25 and fractioned
by a stepwise gradient of ammonium carbonate buffer up to 0.4 M
to purify the reduced NADPH/2H and lyophilization. Purity was
determined by NMR spectroscopy.
as mentioned above. Products of lauric acid hydroxylation sepa-
rated on the same column, with a different temperature gradient
as follows (100 °C, 1 min, 15 °C minꢁ1 to 220 °C, 70 °C minꢁ1 to
290 °C, 2 min hold) and detected at m/z 117, 131, 145, 345 [22].
Apparent Michaelis–Menten constants Km and Vmax, under linear
conditions of time and protein, were derived after nonlinear
regression analysis of the kinetic data using a simple enzyme
kinetics model in SigmaPlot 2004 windows version 9.0 (Systat Soft-
ware, Chicago, IL). Kinetic data is reported as the mean SD
(computer generated).
2.2. Cloning and expression of WT and mutants of P450BM-3
The mutations F87A, T268A and F87A/T268A were introduced
using a QuickChange Mutagenesis kit (Stratagene, La Jolla, CA)
and mutations were confirmed through sequencing the entire gene.
The truncated P450BMP (P450-domain only) and P450BMP T268A
domain, and P450BM-3 reductase domain were generated accord-
ing to [14,15]. P450BM-3 WT and mutants were expressed and
purified by established methods [14–16]. Protein was expressed
in E. coli BL21 DE3 cells in Terrific broth cultures (37 °C, 160 rpm)
supplemented with 1 mM IPTG, 0.5 mM d-aminolevulenic acid
and ampicillin (100 mg Lꢁ1) after 4 h for induction. Cells were
harvested after 48 h by centrifugation. Following cell rupture using
two passes through the micro fluidization machine (Micro DeBEE,
B.E.E. Int., Easton, MA), the cell preparation was centrifuged for
1 h at 37 K rpm (Sorvall, Ti 50.2). The supernatant was directly
loaded onto an affinity 20,50-ADP-sepharose 4B (General electric,
Uppsala, SE) pre-equilibrated with buffer A (containing 100 mM
potassium phosphate, pH 7.4, and 0.5 mM DTT). The column was
washed with buffer B (containing 500 mM potassium phosphate,
pH 7.4, and 0.5 mM DTT) for at least five column volumes. The pro-
tein was subsequently eluted with buffer A, containing 10 mM
NADP, and dialyzed against buffer A (without DTT).
2.5. Solvent isotope effect and incorporation of deuterium
For the measurement of the solvent isotope effect, the enzyme
solution was repeatedly dialyzed (30 kDa cutoff membrane)
against phosphate buffer in D2O with the correction of changes
to pH due to deuterium. Deuterium incorporation into MBA was
measured by replacing NADPH with the pro-R and pro-S NADP2H
using the assay method above and followed the detection of m/z
122 for the mass increase due to the incorporation of deuterium
at the benzylic position.
2.6. Assays for mechanistic characterization
Inhibition of the MBA-reduction by carbon monoxide or imidaz-
ole was measured by saturating the assay buffer with carbon mon-
oxide or adding 300 lM imidazole respectively prior to initiating
the assay as mentioned under kinetic assays. Reversibility of the
MBA-reduction was measured by adding 1 mM NADP+ and
300 lM methoxy-benzalcohol instead of MBA and NADPH into the
assay solution and testing for the formation of NADPH spectropho-
tometrically at 340 nm and MBA via gas chromatography (m/z 137).
2.3. Characterization of P450BM-3 WT and variants
2.7. Binding spectra
P450 CO-difference spectra, gel electrophoresis, pyridine hemo-
chromogen analysis and Lowry protein determination assay were
used to evaluate CYP-enzyme quality and quantity [17–20]. FMN
and FAD content was measured fluorometrically as previously de-
scribed [21] and quantified by standard addition method (addition
of flavin standards to the sample). Cytochrome c reduction activity
For substrate binding experiments, the enzyme was dissolved in
50 mM Tris buffer pH 7.4 and the substrate added (in DMSO) to the
appropriate final concentration (10 nM–100 lM). The increase of
high spin state measured at 390 nm over the decrease in low spin
state (420 nm) was measured against a blank with buffer, enzyme,
and adjusted to the same DMSO concentration (difference spectra).
was measured in 50 mM KPi pH 7.4 with the addition of 100
lM
cytochrome c, 100 M NADPH and 1 nM enzyme, and the steepest
l
slope of the assay was subtracted from a blank measurement [16].
2.4. Enzymatic assays (MBA reduction and lauric acid oxidation)
For substrate reduction assays, enzyme solutions were incu-
3. Results and discussion
3.1. Activity of aldehyde reduction in P450BM-3
bated in phosphate buffer (50 mM KPi, pH 7.4) with 100
strate (dissolved in acetonitrile, final concentration 1%) and after
2 min pre-incubation initiated with NADPH (final concentration
l
M sub-
During the expression of P450BM-3 and its variants, the incor-
poration of heme and/or both flavin prosthetic groups can be re-
duced, severely altering activity. A quality check of the proteins
indicated that P450BM-3 F87A/T268A, P450BM-3 WT and BMR
showed similar redox cofactor incorporation into the apoprotein
(heme, FAD and FMN) with similar ratios of flavins to heme be-
tween proteins (Table 1). Cytochrome c reduction was similar in
P450BM-3 WT and BMR but increased about 25% in P450BM-3
F87A/T268A.
As a model substrate to characterize the aldehyde reduction, p-
methoxy benzaldehyde (MBA) was used. Aldehyde reduction to the
corresponding alcohol was reproducible among several protein
batches and the highest turnover achieved was 180 minꢁ1 mea-
sured for P450BM-3 F87A/T268A variant in one expression. 4-
Methoxybenzoic acid was not observed excluding the possibility
of disproportionation as a mechanism to generate the methoxy
benzyl alcohol from two aldehyde molecules, and no reduction
was demonstrated in control assays in absence of NADPH or
protein.
of 1 mM in 200
were extracted once with an equal volume of ethyl acetate, centri-
fuged at 10,000 rpm, and 80 L of the organic layer derivatized with
20 L of BSTFA for 4 h at 70 °C. One microliters of sample of silated
lL). After 5 min incubation at 37 °C, the incubations
l
l
solutions was injected onto a GC-column DB-1 (15 m, 0.2 mm, Agi-
lent Technol., Santa Clara, CA) installed in a gas chromatograph (HP
GC-17A) equipped with a mass selective detector (GCMS-QP5050),
compounds separated using a temperature gradient program
(40 °C, 1 min, 70 °C minꢁ1 to 160 °C, 15 °C minꢁ1 to 220 °C,
70 °C minꢁ1–290 °C, 2 min hold). Substrate and internal standard
(2-hydroxymethyl naphthalene 20 mg Lꢁ1) were detected at m/z
121 (122 for deuterium incorporation) and 141, respectively.
For the determination of lauric acid oxidation, 0.1
lM enzyme
was incubated under the same conditions as above with 100
lM
lauric acid and extracted with 3 ꢂ 2 mL ethyl acetate. Combined
extracts were evaporated to dryness, resuspended and derivatized

466
R. Kaspera et al. / Biochemical and Biophysical Research Communications 418 (2012) 464–468
Table 1
Kinetics of p-methoxy-benzaldehyde reduction by WT and variants.
Ratio heme:FAD
Ratio FAD:FMN
Cytochrome c reduction per FAD (minꢁ1
)
Km
(lM)
kcat pmol (pmol P450 min)ꢁ1
WT
F87A T268A
BMR domain
1.05
1.03
N/A
0.75
0.78
0.74
2800 500
3800 400
2700 200
150 20
200 20
250 30
23
38
9.9 0.3
1
2
N/A not applicable.
A qualitative assessment of reduction activity towards different
probe substrates demonstrated that P450BM-3 F87A/T268A was
also able to reduce similar aromatic aldehydes with almost equal
activity including naphthalene-2-carbaldehyde, 1,2,3,4-tetrahy-
dro-naphthalene-2-carbaldhyde, 1-H-indole-3-carbaldehyde, and
benzaldehyde, whereas activity for P450BM-3 WT and BMR were
low (data not shown).
variants were able to enhance reductions. In contrast, activity of
lauric acid oxidation was almost completely abolished in
P450BM-3 F87A/T268A (5% residual activity) in comparison to
P450BM-3 WT, while F87A and T268A retained about 20–80% oxi-
dative activity. Thus, a reduced lauric acid oxidation might corre-
spond to an increase rate of aldehyde reduction.
3.3. Probing the aldehyde reduction mechanism
3.2. Kinetic evaluation of reduction in P450BM-3 variants
The reduction mechanism was investigated by several initial
experiments. To transport electrons to the heme active site of
P450BM-3, binding of a reductive equivalent such as NADPH and
transfer of the pro-R-hydride onto FAD takes place [26]. A proton
coupled electron transfer subsequently reduces the second cofac-
tor FMN to a semiquinone or further hydroquinone within the
FMN binding domain, which only moves towards the P450 domain
and reduces the heme center in the first case but inhibits the trans-
fer to the heme in the latter [1]. As previously reported, catalytic
activity for cytochrome c reduction is about 3000 minꢁ1 for
P450BM-3 [16], consistent with our findings. The possibility that
the hydride transfer from NADPH to FAD within the NADPH bind-
ing domain might not yield 100% efficiency would explain a ‘hy-
dride leaching’. In the case of MBA reduction, about 1% of the
total cytochrome c activity would be transferred to a reducible
substrate for P450BM-3 WT and the F87A T268A variant.
First we determined whether a hydride transfer from pro-R- or
pro-S-NADP2H occurred. Indeed, a deuterium is incorporated from
NADP2H into MBA evidenced by the increase of the signal for the
mass fragment m/z 122 (from 121 fragment) and was large for
pro-R-NADP2H (Fig. 2). This indicates a direct hydride transfer onto
the substrate from NADP2H or potentially reduced flavin. Since the
monitored fragment m/z 121 is derived following the cleavage of
O–Si(CH3)3 and there are no other sites on MBA that can accept a
hydride, this is direct evidence of a hydride transfer to the car-
bonyl. A direct analysis of the product by NMR was not feasible
due to difficulty and low yields in the synthesis of pro-R-NADP2H.
A direct hydride transfer was confirmed in experiments conducted
Kinetic evaluation of MBA-reduction demonstrated saturable
kinetics invoking an enzyme catalyzed reaction (Supplementary
material and Table 1). Several expressed P450BM-3 variants were
additionally evaluated to assess a possible involvement of the sep-
arate domains of P450BM-3, and if the reaction can be enhanced
through particular active site variants. E.g. P450BM-3 T268A is
known to reduce the oxidative metabolism and potentially be in-
volved in the peroxyradical cleavage process [23–25], whereas an
F87A enlarges the active site [23]. Interestingly, the P450BM-3
F87A/T268A double mutant catalyzed the reduction with a twofold
higher kcat than P450BM-3 WT or BMR domain alone (Table 1). The
Km values for all tested enzymes were lowest for P450BM-3 WT,
and highest for BMR domain. To assess the involvement of the
heme and reductase domain separately, additional testing of (a)
P450BM-3 protein variants carrying a single mutation within the
P450 domain (F87 and T268), (b) truncated heme domain variants
(BMP), and (c) BMR separately revealed that P450BM-3 F87A/
T268A was the most active variant at a single concentration of
100 lM (Fig. 1). The truncated domain variants BMP WT and
BMP T268A catalyzed the reduction only in trace amounts, and
combining P450BM-3 WT or BMP T268A with BMR did not result
in similar activities as the naturally fused domains. This indicates
that both heme and reductase domains have to be fused to yield
the most efficient aldehyde reduction activity, and P450 active site
Fig. 2. Mass traces of extraction from MBA reduction using CYP450BM-3 F87A
T268A and (A) 1 mM R-NADP2H, and (B) 1 mM S-NADP2H. R121 shows the mass
trace of fragment of non-deuterated alcohol, R122 depicts the mass trace of
deuterated alcohol. Inserted structure depicts the generation of fragment m/z 121
(122 after deuteration).
Fig. 1. Percentage activity of CYP450BM-3 WT and variants, BMR and combined
enzyme domains (activity of CYP450BM-3 F87A/T268A for p-methoxy benzalde-
hyde (MBA) reduction set to 100%, reaction performed at 100 lM MBA).

R. Kaspera et al. / Biochemical and Biophysical Research Communications 418 (2012) 464–468
467
D2O as solvent (see below) where the rate of reaction was reduced
but no deuterium was incorporated in the product confirming that
none of the protons on the product are solvent exchangeable and
the increase of m/z of 1 (from 121 to 122) when reactions are con-
ducted with R-NADP2H can only occur through a hydride transfer
from NADPH (either directly or via FAD) to the carbonyl of MBA.
Estimating the ratio of mass increase from deuterium incorpora-
tion using the area of m/z 122 minus the naturally occurring iso-
tope peak (area of m/z 122) from non-deuterated alcohol product
generated using non-deuterated NADPH, and normalized over the
total signal intensity (area of m/z 121 + m/z 122) indicates that
91 16% of the area of m/z 122 derived from a hydride transfer
originating from pro-R-NADP2H. Using pro-S-NADP2H, only an esti-
mated 6.0 0.5% of deuterium was incorporated according to this
calculation, indicating the enantioselective abstraction of the pro-
R-deuterium over pro-S as previously described for enzymes of
the NADPH-oxidoreductase family [26]. The m/z 122 mass trace
in Fig. 2B thus derives from the above mentioned isotope peak
The purity of the produced NADP2H enantiomers over NADP was
>95% and the enantiopurity 85% and 94% for pro-R- and pro-S-
NADP2H respectively, according to NMR analysis (Supplementary
material) assuming that the reaction might even yield a higher
percentage of enantioselectivity.
at 450 nm for the absorption heme–CO complex. Using P450BM-3
F87A/T268A after saturation of the assay buffer with carbon mon-
oxide, activity of MBA reduction dropped to 30% of initial activity
(Fig. 3), indicating that complexing ferrous heme with CO some-
how interferes with the hydride transfer. Interestingly, 300 lM
imidazole did not inhibit the reduction possibly indicating the
importance of the heme spin state and reducibility. Aldehyde
reduction was not reversible, as the incubation of MBA with NADP
did not produce detectable amounts of aldehyde or NADPH.
Fourth, we conducted ligand binding experiments to test for
aldehyde binding to the heme. The change of spin state was very
low in P450BM-3 F87A/T268A and P450BM-3 WT and MBA caused
an almost irrelevant reverse type I spectra with an estimated KD of
0.11
lM. Ligand binding for P450BM-3 F87A/T268A and P450BM-3
WT was similar for lauric acid (type I, KD 230
l
M and 190 M,
l
respectively) leading to a significant shift in low to high spin heme
iron. No significant heme spin state shift upon substrate binding
indicates essentially no MBA binding in the P450 domain. This
observation was substantiated by a high Km for MBA for all pro-
teins tested which was interestingly also much higher compared
to substrates such as cytochrome c not binding in the heme do-
main [28]. Nevertheless, saturable binding kinetics indicate an ac-
tual substrate binding site, presumably close to the NADPH binding
site or flavins which is necessary for hydride transfer.
Second, we determined the effect of D2O on the aldehyde reduc-
tion possibly affecting protein–protein interactions and proton
transfer mechanisms. Exchanging the aqueous buffer with D2O
based potassium phosphate buffer decreased MBA reduction by
about 60% in P450BM-3 F87A/T268A (Fig. 3) but, and most impor-
tantly, without introducing a deuterium into the alcohol product.
This could indicate that either protein–protein, domain interac-
tions or proton transfer mechanisms involved in the reduction of
flavins [27] were slowed down. Consequently, a reduction via the
FMN hydroquinone cannot be excluded, as the mechanism of a hy-
dride transfer is identical for both flavins. Interestingly, the high
catalytic activity for cytochrome c reduction in the P450BM-3
F87A T268A variant in comparison to the WT seem to correlate
with a high activity of a hydride transfer, which could be explained
by a possible interference of the flavin semiquinone/hydroquinone
state during reduction. A compromised heme one electron reduc-
tion and substrate oxidation can be caused by NADPH, if a flavin
reduction occurs prior to substrate addition [3]. A similar ‘feed-
back’ in the redox cascade might have occurred during the reduc-
tion and CO binding to the heme domain, favoring a hydride
‘leaching’ to an easily reducible substrate.
Besides the hydride transfer from NADPH, an alternative possi-
ble mechanism could be speculated similar to the morpholine
reductase directly transferring a hydride from a piperazine moiety
of the flavins hydroquinone (FADH2 or FMNH2 in an ‘overreduced’
P450BM-3 [5]) to a reducible carbonyl [29] (Fig. 4). Previous re-
ports propose a mechanism involving a reduction of aldehydes
such as hydroxy nonenal by CYP450-enzymes through a radical
formation and single electron reduction of the radical intermediate
via the iron(II) species [11,12]. As the BMR domain alone was able
to perform the reduction of MBA and the almost complete hydride
transfer from pro-R-NADPH demonstrated a direct reduction by
this domain, the described reaction seems unrelated to the radical
mechanism. An increase in activity through an additional radical
mechanism using P450BM-3 WT and mutants consisting of both
P450 and reductase domain might be excluded and could possibly
be caused by protein–protein interactions or a proton coupled
electron transfer as evidenced by decreased turnover in D2O sol-
vent which is suggestive of proton movement.
The reduction of aldehydes by P450BM-3 through a direct hy-
dride transfer has not been described previously and speaks to
the versatility of this enzyme in facilitating oxidations and reduc-
tions. Interestingly, this reduction seems to be augmented in the
fused P450 domains through a potential protein domain interac-
tion probably based on redox kinetics or allosteric interactions.
Third, we tested the involvement of the heme domain of
P450BM-3 and reversibility of the aldehyde reduction. Reduction
of heme can be measured by the saturation of heme with carbon
monoxide and subsequent reduction via NADPH to form the soret
Fig. 3. Percentage activity of for p-methoxy benzaldehyde (MBA) reduction for
CYP450BM-3 WT and CYP450BM-3 F87A/T268A using D2O as a solvent and further
testing inhibition by carbon monoxide and imidazole (control activity set to 100%,
Fig. 4. Possible mechanism for the reduction of an aldehyde substrate by a hydride
reaction performed at 100
l
M MBA).
transfer from NADPH (1) or flavin hydroquinone (2).

468
R. Kaspera et al. / Biochemical and Biophysical Research Communications 418 (2012) 464–468
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Investigations are ongoing to determine, the importance of the
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bbrc.2012.01.040.
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